Ling Yang
email: ling.yang@ctsu.ox.ac.uk

Maigeng Zhou

Margaret Smith

Gonghuan Yang

Richard Peto

Jun Wang

Jillian Boreham

Yisong Hu

Zhengming Chen

Body mass index and COPD-related mortality: A nationally representative prospective study of 220 000 men in China

Keywords: body mass index, smoking, prospective study, COPD, mortality reference, tables, footnotes, figure legends): 3087 body mass index, smoking, prospective study, COPD, mortality

Background: Low body mass index (BMI) is associated with chronic obstructive pulmonary disease (COPD) in populations where many are overweight. Substantial uncertainty remains about the relationship in populations with lower mean BMI levels, and about the relevance to it of the effects of smoking.

Methods:

A nationally representative prospective cohort study included 221,194 Chinese men aged 40-79 years in 1990-91, who were followed up for 15 years. Hazard ratios of COPD-related death relating to baseline BMI were adjusted for age, smoking, drinking and other factors. To reduce reverse causality, main analyses excluded all men with prior history of any respiratory diseases or abnormal lung function at baseline, leaving 2,960 COPD-related deaths (16% of total deaths).

Results:

The mean baseline BMI was 21.7 kg/m 2 . There was a highly significant inverse association between BMI and COPD-related mortality among men without any apparent impairment of lung function. Approximately 90% of men had a baseline BMI<25 kg/m 2 , and among them, 5 kg/m 2 lower BMI was associated with 31% (95% CI: 18%-45%) higher COPD-related mortality. The excess risk persisted after restricting the analysis to never-smokers or excluding the first 5 years of follow-up.

Conclusions:

Low BMI is associated with increased risk of COPD mortality in a relatively lean adult male population in China where COPD is one of the most common causes of death.

BACKGROUND

Chronic obstructive pulmonary disease (COPD) is a leading cause of mortality and disability world-wide, [START_REF] Viegi | Epidemiology of chronic obstructive pulmonary disease (COPD)[END_REF] with an estimate of over 200 million affected individuals and about 3 million annual deaths. 2 In China, COPD causes about 1.3 million deaths each year 3 and the prevalence of spirometric-detected COPD in people aged 40 years or above has been reported to be 8%, with three-quarters of those affected having clinically manifest disease. [START_REF] Zhong | Prevalence of chronic obstructive pulmonary disease in China: a large, population-based survey[END_REF] Despite this high disease burden, epidemiological evidence about aetiology of COPD in China is limited. [START_REF] Chan-Yeung | The burden and impact of COPD in Asia and Africa[END_REF] In Western populations, where cigarette smoking has persisted for many decades, smoking is responsible for most of COPD deaths. 6;7 In China, the situation is quite different, with only about half of the male COPD deaths being attributable to smoking [START_REF] Liu | Emerging tobacco hazards in China: 1. Retrospective proportional mortality study of one million deaths[END_REF] , and the disease rate is high even among individuals who have never smoked, especially in certain inland rural regions. 4;9;10 It has been suggested that, in addition to smoking, other factors such as some types of indoor air pollution, chronic infection and malnutrition may also contribute significantly to risk of the disease. 1;11-14 Several hospital-based studies have shown that low body mass index (BMI, weight in kilograms divided by the square of the height in meters) is an independent prognostic factor for both long-term survival following diagnosis of COPD [START_REF] Vestbo | Body mass, fat-free body mass, and prognosis in patients with chronic obstructive pulmonary disease from a random population sample: findings from the Copenhagen City Heart Study[END_REF][START_REF] Menezes | Prevalence of chronic obstructive pulmonary disease and associated factors: the PLATINO Study in Sao Paulo, Brazil[END_REF][START_REF] Landbo | Prognostic value of nutritional status in chronic obstructive pulmonary disease[END_REF][START_REF] Montes De Oca | Chronic obstructive pulmonary disease and body mass index in five Latin America cities: The PLATINO study[END_REF][START_REF] Schols | Weight loss is a reversible factor in the prognosis of chronic obstructive pulmonary disease[END_REF][START_REF] Prescott | Prognostic value of weight change in chronic obstructive pulmonary disease: results from the Copenhagen City Heart Study[END_REF] and disease severity, 21 . In cross-sectional studies, COPD is more prevalent among lean people. 6;22-25 Recent data from large prospective cohort studies have also shown that low BMI is associated with COPD. However, most such studies have been in Western populations where the prevalence of COPD is relatively low, especially among non-smokers, and few have low BMI persistently throughout childhood and adult life. [START_REF] Watson | Predictors of lung function and its decline in mild to moderate COPD in association with gender: results from the Euroscop study[END_REF][START_REF] Harik-Khan | Body mass index and the risk of COPD[END_REF][START_REF] Pischon | General and abdominal adiposity and risk of death in Europe[END_REF][START_REF]Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies[END_REF] Furthermore, most such studies have included small number of deaths and did not control appropriately for

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smoking or for the effects of pre-existing disease on BMI (the reverse causality). [START_REF]Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies[END_REF] We report a 15-year prospective study of the association between BMI and COPD mortality in a nationally representative cohort of 221,000 men in China recruited during 1990-91, including 148,000 current smokers and 59,000 never-smokers.

MATERIALS AND METHODS

Baseline survey

Details of the study design, participants and baseline survey methods have been reported previously. 30;31 Briefly, the study involves 45 areas (23 urban and 22 rural) which were randomly selected from China's 145 Disease Surveillance Points (DSPs).

The DSP system was established in the mid-1980s to provide a nationally representative sample of mortality statistics for the entire country. 32;33 A typical surveillance point covers a defined population of about 50,000-100,000 residents in 4-8 geographically defined units (ie, urban street committees or clusters of rural villages).

During 1990-91, all men aged 40 years or over from two or three randomly selected units within each of the 45 areas were invited to participate in the survey, and about 80% of invitees attended. In the survey clinics set up for the study, trained health workers administered a standardized questionnaire, which included education, occupation, tobacco, alcohol, dietary pattern, exposure to indoor air pollution, selfreported medical history and health status. Physical measurements included blood pressure, height, weight and peak expiratory flow rates. Waist and hip circumferences and subsequent weight changes were not measured.

Follow-up for cause-specific mortality

The vital status of each study participant was monitored regularly by DSP staff through their death registries previously established in these areas. In addition, active Causes of death were sought chiefly from official death certificates, supplemented, if necessary, by review of medical records. [START_REF] Yang | Exploring adult mortality in China: levels patterns and causes[END_REF] The underlying cause of each death was coded centrally by DSP staff in Beijing, without knowledge of the baseline information, using the ninth revision of the International Classification of Disease (ICD-9). In the few deaths without any recent medical attention, standard procedures were used by local DSP staff to determine the probable cause from symptoms or signs described by family members. [START_REF] Yang | Exploring adult mortality in China: levels patterns and causes[END_REF] COPD-related death was defined as death from chronic bronchitis, emphysema, asthma, bronchiectasis, other chronic obstructive pulmonary disease, or pulmonary heart disease (ICD-9 codes 415-417 and 490-496).

Since it is often difficult to assign an underlying cause reliably for death in old age, all analyses were restricted to deaths occurring between age 40-79, with censoring when

Statistical analysis

Cox proportional hazards models were used to calculate hazard ratios (HRs), with BMI as the exposure variable and COPD-related death as the outcome. All analyses were stratified by area (45 strata) and by 5-year age group at risk, and were adjusted simultaneously for education, tobacco (never, former, current), alcohol (at least weekly/not), regular exposure to indoor domestic or occupational air pollution from coal or biomass (yes/no), and consumption of certain food stuffs (times/week), such as meat, vegetable and fruit. Baseline BMI was divided into five categories, with cut-off points at The 95% confidence interval (CI) for each log HR was estimated using the 'floating absolute risk' method. [START_REF] Easton | Floating absolute risk: an alternative to relative risk in survival and case-control analysis avoiding an arbitrary reference group[END_REF] This facilitates many different comparisons and tests for trend between different BMI categories, rather than just pair-wise comparisons between one arbitrarily chosen reference group and each of the other categories. Trend tests were conducted treating BMI as a continuous variable in the Cox model. The analyses were done in SAS version 9.1 under Windows XP.

RESULTS

For the 221,194 participants (mean baseline age of 54.3 years), overall mean BMI was 21.7 (SD 2.7) kg/m 2 , with 9.2% being underweight (BMI <18.5 kg/m 2 ), and 11.1% being either overweight (BMI 25 to <30 kg/m 2 , 10.3%) or obese (BMI ≥30 kg/m 2 , 0.8%). The mean age-adjusted BMI was higher among urban than among rural men (23.1 vs 21.2 kg/m 2 ). After adjusting for area and age, it was strongly positively related to blood pressure (table 1), with similar gradients for smokers and never-smokers (data not shown). Men with high BMI were slightly better educated, and were somewhat more likely to drink alcohol. The prevalence of smoking was strongly inversely associated with BMI, but among current smokers, the mean number of cigarette smoked per day was not associated with BMI (which implies that, among smokers, the dose of tobacco per kilogram of body weight was inversely related to BMI). Men with low BMI were more likely to report a prior history of disease at baseline. (table 1)

During the 15-year follow-up, 40,907 died before age 80, of which 8,769 (21.4%) were due to COPD-related conditions. Of the remainder, 34,165 (1% per annum) were lost to follow-up, mainly because of demolition of neighborhood for redevelopment. Those lost

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did not differ significantly from those not in the main baseline characteristics such as age, SBP, BMI, prevalence of ever-smoking. [START_REF] Chen | Body mass index and mortality from ischaemic heart disease in a lean population: 10 year prospective study of 220,000 adult men[END_REF] The standardized COPD mortality rate was more than twice as high among men with a history of respiratory disease as among men without it (540 vs 203 per 100,000). It was also about twice as high among rural men as among urban men (290 vs 156), and higher among ever-smokers than among never-smokers (265 vs. 223). Overall there was a highly significant inverse association between BMI and COPD-related mortality, with the death rate in the lowest BMI group (<18.5 kg/m 2 ) being 2.5 fold that in the highest group (≥25 kg/m 2 ) (table 1).

In the study, the peak expiratory flow rate was strongly inversely associated with the COPD-related death (figure 1). However, the inverse association between BMI and COPD-related death was particularly steep among men with impaired lung function at baseline, defined as having pre-existing respiratory diseases (i.e. TB, chronic bronchitis, asthma, emphysema or pulmonary heart disease), or self-reported chronic respiratory symptoms (ie cough or shortness of breath) or being in the lowest quintile of age-and height-adjusted peak expiratory flow rate) (figure 2). Among these participants, each 5 kg/m 2 lower baseline BMI was associated with 118% (95%CI: 104%-134%) higher risk of COPD-related death for BMI <25 kg/m 2 ), while in men without impaired lung function, the increased risk was much less extreme (30% [17%-44%]). The exclusion of the first 5 years of follow-up did not materially alter the relationships (94% [76%-113%] higher risk per each 5 kg/m 2 lower BMI for men with impaired lung function versus 34% [17%-53%] higher risk for men without it). To help minimize the effects of reverse causality, those with impaired lung function at baseline were excluded from subsequent analyses.

Among men without any apparent impairment of lung function, the relationship of BMI with COPD mortality varies little at different periods of follow-up (p=0.4 for heterogeneity

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among the three follow-up periods, figure 3). Moreover, further exclusion of the small number of individuals who reported poor health status at baseline did not materially change the association (data not shown). The inverse association between BMI and COPD-related mortality was slightly more extreme in never-smokers than in eversmokers (figure 4). Among ever-smokers, each 5 kg/m 2 lower BMI was associated with 24% (10%-40%) higher risk at BMI<25 kg/m 2 , whereas among never-smokers, each 5 kg/m 2 lower BMI was associated with 49% (22%-82%) higher risk. After further exclusion of the first 5 years of follow-up, the association was not altered, with each 5 kg/m 2 lower BMI associated with 29% (11%-51%) and 52% (15%-99%) higher risks among ever-and never-smokers respectively.

Figure 5 shows the association of BMI with COPD-related death separately for different age groups. The inverse association appeared to be somewhat steeper at age <70 than at age 70-79 years (p<0.05 for heterogeneity test). Among men with BMI<25 kg/m 2 , there was little evidence that the strength of the association was significantly modified by other factors such as locality, level of education, alcohol drinking or exposure to indoor air-pollution (figure 6).

In the study, COPD accounts of 87% of all respiratory deaths, with a further 595 (6%) deaths from TB. For TB mortality, a significant inverse association with BMI was also seen, with HRs of 2.6, 1.6, 1.0, 0.7 and 0.3 for men with BMI <18.5, 18.5-20, 20-22.5, 22.5 and >=25 kg/m 2 respectively. At BMI <25 kg/m 2 , each 5 kg/m 2 lower BMI was associated with 237% (95%CI: 170%-319%) higher TB mortality.

DISCUSSION

F o r R e v i e w O n l y 10
This is one of the largest prospective studies of the relationship between BMI and COPD-related mortality in a relatively lean population, and it involved a nationally representative population of Chinese men with nearly 9,000 COPD-related deaths. In this population, approximately 90% of men had baseline BMI<25 kg/m 2 (as opposed to less than 50% in many Western countries 35;36 ), and among them there was a highly significant inverse relationship between BMI and COPD-related mortality with each 5 kg/m 2 lower BMI associated with 30% higher mortality. The association was largely independent of age, smoking and other factors, and the extent to which the association could be biased by reverse causality was greatly limited by exclusion of all who had evidence of impaired lung function at baseline.

Several prospective studies both in Western and in East Asian populations have reported an inverse association between BMI and COPD. 27-29;37 There were differences in the strength of the relationship between these studies, which may be partly due to difference in the completeness of control for reverse causality by prior disease and for confounding by smoking. Moreover, most previous studies have not individually recorded large numbers of COPD cases, especially at lower levels of BMI, so the shape of the association between BMI and COPD may be particularly susceptible to the play of chance. For example, in the recently published European Prospective Investigation into Cancer and Nutrition (EPIC) among 360,000 participants, only 637 respiratory deaths (4% of total deaths) were recorded after 10 years of follow-up. [START_REF] Pischon | General and abdominal adiposity and risk of death in Europe[END_REF] In that study, there was a strong inverse association between baseline BMI and respiratory mortality, with the risk among men with BMI<18.5 kg/m 2 being 6.5 times that among men with BMI 23.5-25.0 kg/m 2 . However, it was not clear to what extent the excess risk at low BMI could be attributed to reverse causality, for the analysis did not exclude those with prior respiratory disease. In a recently published collaborative meta-analysis of BMI and cause-specific mortality among 900,000 individuals in 57 prospective studies (the Prospective Studies Collaboration (PSC), mostly in Western Europe and North America), [START_REF]Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies[END_REF] COPD mortality (1,958 deaths) was strongly inversely associated with BMI, with each 5 kg/m 2 lower BMI associated with a four-fold increased risk within the BMI range of 15-25 kg/m 2 . Above BMI 25 kg/m 2 , there appeared to be a weakly positive relationship between BMI and COPD mortality. In the PSC, COPD was defined similarly as in the present study, with the exception of pulmonary heart disease, but it could not exclude people with COPD at baseline, nor those with abnormal lung function.

Consequently, reverse causality was controlled for mainly by excluding the early period of follow-up, [START_REF]Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies[END_REF] which may help to explain why the inverse association with COPD mortality was much more extreme in PSC than in this study. On the other hand, smoking is much more strongly associated with COPD in Western populations than in Chinese, [START_REF] Liu | Emerging tobacco hazards in China: 1. Retrospective proportional mortality study of one million deaths[END_REF] and so is the relationship between smoking and BMI. [START_REF]Body-mass index and cause-specific mortality in 900 000 adults: collaborative analyses of 57 prospective studies[END_REF] This suggests that confounding by smoking may be more substantial in the Western studies than in the present study of Chinese, and failure to control it effectively could potentially inflate the excess risk associated with low BMI.

The overall findings of the present study are generally consistent with limited data from other prospective studies in Asian populations where the rate of COPD is relatively high and many people have relatively low BMI persistently throughout childhood and adult life. [START_REF] Jee | Body-mass index and mortality in Korean men and women[END_REF][START_REF] Reilly | Risk factors for chronic obstructive pulmonary disease mortality in Chinese adults[END_REF][START_REF] Sauvaget | Body mass index, weight change and mortality risk in a prospective study in India[END_REF] In a large prospective study of 170,000 adult Chinese, [START_REF] Reilly | Risk factors for chronic obstructive pulmonary disease mortality in Chinese adults[END_REF] 10.8% men and 12.5% women were underweight at baseline, and there were 2,470 COPD-related deaths after about 8 years of follow-up. As in the present study, smoking was found to be only weakly associated with COPD mortality, with RR of 1.2 for men who had smoked for at least 20 pack-years compared with never-smokers. Among men, BMI was inversely associated with COPD-related mortality, with RR of 2.7 (95% CI: 2.3-3.0) among

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underweight men compared with those who were not. However, no information was available in that study about history of pre-existing disease or measured lung function at baseline, so the control for potential reverse causality bias was limited chiefly to the exclusion of the first few years of follow-up.

In the present study, potential biases related to reverse causality from prior disease and confounding by smoking have been dealt with rigorously. A reliable diagnosis of COPD is usually based on a typical history of persistent progressive symptoms, existence of an appropriate risk factor (such as smoking), and a confirmatory spirometric test. [START_REF] Calverley | Chronic obstructive pulmonary disease[END_REF] In China, only about a third of patients with spirometric evidence of COPD had ever been clinically diagnosed, and less than 10% had been tested with spirometry. 4;42 Moreover, patients with COPD are often likely to die from other causes, with less than half having COPD certified as the underlying cause of death. [START_REF] Reilly | Risk factors for chronic obstructive pulmonary disease mortality in Chinese adults[END_REF] To help reduce potential underreporting of COPD as cause of death, we used COPD-related mortality as our main endpoint for analysis, which included not only COPD deaths, but also those with asthma and pulmonary heart disease as the underlying causes of death. This definition was compatible with that used in another large COPD-related study in China [START_REF] Reilly | Risk factors for chronic obstructive pulmonary disease mortality in Chinese adults[END_REF] and in PSC 27 . Due to lack of spirometry measurement at baseline, we used the measured peak flow rate as an indicator of pulmonary function. Though not ideal, it should have helped to identify effectively undiagnosed cases of COPD at baseline. [START_REF] Pauwels | Burden and clinical features of chronic obstructive pulmonary disease (COPD)[END_REF] In addition to excluding those with any prior respiratory disease or abnormal lung function at baseline, data were also examined separately among ever-and never-smokers and by different periods of follow-up. The strong inverse relationship of BMI to risk of COPD-related death persisted even after excluding the first 10 years of follow-up, and was present not only among smokers but also among never-smokers. So, the relationship is not likely to The inverse relationship between BMI and risk of COPD-related death could represent a combination of various factors. COPD can lead to weight loss over a prolonged period of time and many of the patients with COPD, despite exclusion of those with prior disease, with impaired lung function, or first 5 years of follow-up, may still not be properly identified, so it is still possible that the inverse association observed could still have been mainly due to reverse causality. On the other hand, some close correlate of low BMI, such as malnutrition, could itself cause COPD, or increase its progression and, consequently, mortality. [START_REF] Schwartz | Malnutrition in chronic obstructive pulmonary disease[END_REF] Clinical-based studies showed that low BMI is an independent prognostic factor of poor long-term survival following diagnosis of COPD. 19;45 There is also evidence that low body weight could predispose to an increased risk of chronic infection such as pulmonary TB, 46;47 a disease which was also shown in this study to be strongly inversely related to BMI. If chronic respiratory infections also play a role in the aetiology of COPD, [START_REF] Chapman | Epidemiology and costs of chronic obstructive pulmonary disease[END_REF] it may help to understand the possible biological mechanism underlying the inverse relationship between BMI and COPD death. Newly established large blood-based prospective studies in China [START_REF] Chen | Cohort profile: the Kadoorie Study of Chronic Disease in China (KSCDC)[END_REF] will eventually provide good evidence on it. It has been suggested that other measures of body composition (eg waist and hip ratio, percentage body fat) 15;25;50-52 or instrumental variable like offspring BMI [START_REF] Smith | The association between BMI and mortality using offspring BMI as an indicator of own BMI: large intergenerational mortality study[END_REF] might provide better assessment for disease association, and that low physical activity may also help to identify those with underlying disease that generate the association with COPD. However, no such measurements were available in the present study.

The recent large increase in cigarette consumption [START_REF] Zhang | The impact of tobacco on lung health in China[END_REF] in China may eventually lead to a substantial increase in the risk of COPD in the population, especially among men. 4;42;55 On the other hand, if low BMI were causally associated with increased risk of COPD through mechanisms yet to be elucidated, then the recent rise in mean BMI in the population as a result of improved living standards could potentially lead to decreased risk of COPD in the future. This may counterbalance to some extent the likely increase in the disease rate from increased exposure to smoking, even though the extent and time scale of these changes needs to be closely monitored in future studies.

  status was done annually through local residential committees.

  men reached 80 years of age, died from other causes or moved away from the original study area during follow-up. This report is based on follow-up data to 1,1,2006 among the 221,194 men who were aged 40-79 years and had baseline BMI in the range of 10-50 kg/m 2 .

  20, 22.5 and 25 kg/m 2 . The HR for COPD-related mortality was calculated for each BMI category, with the BMI category of 20.0-22.5 kg/m 2 as the reference group.

  by chance, by prior disease affecting BMI at baseline, or by residual confounding by smoking. It may, however, not necessarily be causal.

Figure 1 .

 1 Figures Legends

17 ( 4 ) 4 .( 5 ) 5 .( 6 )

 1744556 Figure Hazard ratios for death from COPD-related diseases versus baseline BMI among men aged 40-79 with no prior respiratory disease or impaired lung function at baseline, in those who were (a) Ever-smokers and (b) Life-long non-smokers. Conventions as in Figures 1 and 2. Figure Hazard ratios for death from COPD-related diseases versus baseline BMI among men with no prior respiratory disease or impaired lung function at baseline, by age at risk. Conventions as in Figures 1 and 2.Figure 6. Hazard ratios for death from COPD-related diseases per 5 kg/m 2 lower BMI among men with baseline BMI <25.0 kg/m 2 and with no prior respiratory disease or impaired lung function at baseline. Each closed square represents a hazard ratio (HR) with the area inversely proportional to the variance of log HR. The dotted vertical line indicates the overall HR, and open diamonds indicates it and its 95% CI.

Figure 1 F

 1 Figure 1

Table 1 . Baseline characteristics of participants and standardized mortality rate from COPD-related disease by BMI category, all men aged 40-79 years at baseline BMI Categories (kg/m 2 ) Baseline characteristics * All <18.5 18.5-19.9 20-22.4 22.5-24.9 ≥25

 1

	Total population	221194	20338	37748 89079	49418 24611
	Mean BMI (kg/m 2 )	21.7	17.6	19.4	21.3	23.5	26.9
	Mean age (years)	54.3	59.0	55.4	53.4	53.1	54.4
	Living in urban area (%)	27.2	17.9	15.5	19.3	35.6	64.4
	Mean weight (kg)	59.0	48.4	53.0	57.8	63.4	72.3
	Mean height (cm)	164.5	165.5	165.1 164.7	163.9	163.5
	Mean SBP (mm Hg)	124.1	119.4	121.3 123.4	125.8	131.2
	Mean peak flow (l/min.)	395.2	360.8	381.9 397.7	406.8	412.1
	Education ≥6 years (%)	33.1	32.8	31.4	32.3	34.3	36.6
	Smoking status (%)
	Ex-smokers	6.3	6.1	5.2	5.5	6.8	9.8
	Current smokers	67.1	72.3	71.6	68.7	63.7	56.8
	No. of cigarettes per day	11.9	11.6	11.6	11.9	12.1	12.3
	Age started smoking (years)	22.2	22.0	22.1	22.2	22.4	22.7
	Regular alcohol drinking (%)	33.5	30.6	31.9	33.7	34.6	35.1
	Exposure to indoor air-pollution (%)	10.9	11.1	10.9	10.7	11.0	11.1
	Intake of meat (times/week)	3.5	3.3	3.3	3.4	3.6	3.8
	Intake of vegetable (times/week)	13.8	13.7	13.7	13.7	13.8	13.9
	Intake of fruit (times/week)	1.3	1.2	1.2	1.3	1.4	1.6
	History of major chronic disease (%)	19.4	27.7	21.0	17.7	17.2	20.2
	Prior respiratory disease (%)	15.9	24.6	17.9	14.6	13.6	14.5
	Chronic respiratory symptoms (%)	28.4	36.6	30.8	27.0	25.4	29.0
	Self-reported poor health status (%)	7.3	13.5	8.4	6.6	5.5	6.6
	Mortality from COPD †
	No. of deaths	8769	1860	1980	3244	1264	421
	Mortality rate /100,000	255.6	490.9	301.1 235.1	194.5	189.2

Funding and Acknowledgements

This work was supported by the Chinese Ministry of Health; the UK Medical Research Council, British Heart Foundation and Cancer Research UK; the World Bank loan to China; and the Canadian International Development Research Centre. We thank the China DSP staff for their continuing effort and cooperation with long-term follow-up of study participants.

Abbreviations


Björn M Burmann

Ulrich Scheckenhofer

Kristian Schweimer

Paul Rösch
email: roesch@unibt.de

Domain interactions of the transcription:translation coupling factor E. coli NusG are intermolecular and transient

The bacterial transcription factor NusG is suggested to act as a key coupling factor between transcription and translation (Burmann et al. 2010 Science. 328:501-504). and contributes to phage λ mediated antitermination in E. coli that enables read-through of early transcription termination sites. E. coli NusG consists of two structurally and functionally distinct domains that are connected via a flexible linker. The homologous Aquifex aeolicus NusG with a secondary structure that is highly similar to E. coli NusG shows direct interaction between its N-terminal and its C-terminal domain in a domain-swapped dimer. Here we performed NMR paramagnetic relaxation enhancement measurements and identified interdomain interactions that were concentration dependent and thus likely not only weak and transient, but also predominantly intermolecular. This notion of two virtually independent domains in a monomeric protein was supported by 15 N relaxation measurements. Thus, we suggest a regulatory role of NusG interdomain interactions to be highly unlikely.

Introduction

E. coli NusG (N-utilization substance G) is essential for cell viability [START_REF] Downing | Sequence and transcriptional pattern of the essential escherichia coli secE-nusG operon[END_REF] and is found in all known bacteria. The C-terminal domain, CTD, contains a KOW motif that is also found in archaeal [START_REF] Kyrpides | Transcription in archaea[END_REF] and eukaryotic [START_REF] Hartzog | Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in saccharomyces cerevisiae[END_REF] proteins. NusG's exact role in transcription regulation remained obscure until very recently when NusG was identified as the long sought-after coupling factor between transcription and translation in E. coli [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF], in addition to its better known role in the transcription-elongation complex (TEC) where it increases the elongation rate of the RNAP in vivo and in vitro [START_REF] Burova | Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro[END_REF][START_REF] Burns | Combinatorial effects of NusA and NusG on transcription elongation and rho-dependent termination in escherichia coli[END_REF][START_REF] Artsimovitch | Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals[END_REF] by suppression of transcriptional pausing [START_REF] Artsimovitch | Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals[END_REF], which could be shown recently to be due to promoting forward translocation of the RNAP [START_REF] Herbert | E. coli NusG inhibits backtracking and accelerates pause-free transcription by promoting forward translocation of RNA polymerase[END_REF]. NusG, along with the other Nus factors A, B, E (ribosomal protein S10), RNA, and the RNAP, is also a component of the phage λ protein N mediated antitermination [START_REF] Li | NusG, a new escherichia coli elongation factor involved in transcriptional antitermination by the N protein of phage lambda[END_REF][START_REF] Sullivan | Effect of escherichia coli nusG function on lambda N-mediated transcription antitermination[END_REF][START_REF] Mason | Host factor requirements for processive antitermination of transcription and suppression of pausing by the N protein of bacteriophage lambda[END_REF] and the phage HK022 protein Nun mediated termination complex [START_REF] Sullivan | Requirement for E. coli NusG protein in factor-dependent transcription termination[END_REF][START_REF] Burova | Escherichia coli nusG mutations that block transcription termination by coliphage HK022 nun protein[END_REF]. The regulatory functionality of NusG can be attributed to direct interaction with the RNAP [START_REF] Li | NusG, a new escherichia coli elongation factor involved in transcriptional antitermination by the N protein of phage lambda[END_REF][START_REF] Mason | Assembly of transcription elongation complexes containing the N protein of phage lambda and the escherichia coli elongation factors NusA, NusB, NusG, and S10[END_REF]15]. Additionally, NusG is involved in transcription antitermination in ribosomal RNA (rRNA) [START_REF] Torres | In vivo effect of NusB and NusG on rRNA transcription antitermination[END_REF][START_REF] Zhou | Requirement for NusG for transcription antitermination in vivo by the lambda N protein[END_REF] and in termination at certain ρ dependent sites where it recruits ρ [START_REF] Sullivan | Requirement for E. coli NusG protein in factor-dependent transcription termination[END_REF][START_REF] Li | Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription[END_REF][START_REF] Pasman | Regulation of rho-dependent transcription termination by NusG is specific to the escherichia coli elongation complex †[END_REF]. NusG also prevents backtracking of the RNAP [START_REF] Artsimovitch | Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals[END_REF], but detailed information on the final steps of ρ-dependent termination is still lacking [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF]. Direct NusG:ρ interaction was reported [START_REF] Li | Elongation factor NusG interacts with termination factor rho to regulate termination and antitermination of transcription[END_REF][START_REF] Pasman | Regulation of rho-dependent transcription termination by NusG is specific to the escherichia coli elongation complex †[END_REF], and the NusG carboxy-terminal domain (CTD) was identified as the interaction domain [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF]. In addition to its role in transcription, NusG is also involved in translational regulation [START_REF] Zellars | Antiterminator-dependent modulation of transcription elongation rates by NusB and NusG[END_REF], and, again, the NusG CTD was identified as the ribosome interaction domain [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF].

The structure of E. coli NusG [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF] is highly similar to the structures of the homologous Aquifex aeolicus [START_REF] Steiner | Crystal structures of transcription factor NusG in light of its nucleic acid-and protein-binding activities[END_REF][START_REF] Knowlton | A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants[END_REF] and Thermus thermophilus [START_REF] Reay | Structural and sequence comparisons arising from the solution structure of the transcription elongation factor NusG from thermus thermophilus[END_REF] proteins. The A. aeolicus NusG is reported to form a domain-swapped dimer under certain crystallization conditions [START_REF] Knowlton | A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants[END_REF]. However, such a dimer is not observed in a different crystallization setup [START_REF] Steiner | Crystal structures of transcription factor NusG in light of its nucleic acid-and protein-binding activities[END_REF], and ultracentrifugation analysis showed no indication of a non-monomeric form of NusG as an isolated protein [START_REF] Pasman | Regulation of rho-dependent transcription termination by NusG is specific to the escherichia coli elongation complex †[END_REF]. Interestingly, however, the NusG paralogue RfaH is reported to exist in a closed conformation with both domains tightly interacting, and this tight interaction is abolished only upon binding of RfaH to the ops-site (operon polarity suppressor) of the non-template DNA strand, when the RfaH-CTD leaves the binding pocket on the RfaH-NTD, thus providing an RfaH activation step [START_REF] Artsimovitch | The transcriptional regulator RfaH stimulates RNA chain synthesis after recruitment to elongation complexes by the exposed nontemplate DNA strand[END_REF][START_REF] Belogurov | Structural basis for converting a general transcription factor into an operon-specific virulence regulator[END_REF]. Although E. coli NusG and RfaH exhibit the same folding topology of their NTDs [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF][START_REF] Belogurov | Structural basis for converting a general transcription factor into an operon-specific virulence regulator[END_REF](NusG-NTD, PDB-ID: 2K06; RfaH, PDB-ID: 2OUG), their CTDs show reversed topologies: Whereas RfaH-CTD is all α-helical, NusG-CTD exhibits an anti-parallel β-barrel-like structure [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF] (NusG-CTD, PDB-ID: 2JVV).

The area masked on the RfaH-NTD by its CTD consists of several surface-exposed hydrophobic residues [15,[START_REF] Sevostyanova | The elongation factor RfaH and the initiation factor sigma bind to the same site on the transcription elongation complex[END_REF], and surface hydrophobic residues are more numerous in RfaH than in NusG [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF] where they may be involved in additional protein interactions [START_REF] Steiner | Crystal structures of transcription factor NusG in light of its nucleic acid-and protein-binding activities[END_REF]. These lines of results raises the question which properties the common ancestor of these proteins had, in particular whether it was in closed or open form, and whether domain opening provided a regulatory mechanism. To proceed to an answer to this question, among others, we decided to ask whether E. coli NusG domains interact and whether a closed conformation, if only lowly populated, exists.

To address this question and to gain insight into the role of the individual E. coli NusG domains within the TEC, their relative motional behavior and their mutual interactions we employed chemical shift and paramagnetic relaxation enhancement (PRE) perturbation titration as well as relaxation experiments.

Experimental

Cloning, expression, and purification of full-length NusG and the individual domains: Cloning, expression and purificaton was based on published methods [START_REF] Pasman | Regulation of rho-dependent transcription termination by NusG is specific to the escherichia coli elongation complex †[END_REF][START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF]. NusG was cloned via Bpu1102I and NdeI restriction sites into the E. coli expression vector pET11A (Novagen, Madison, WI, USA). E. coli strain BL21(DE3) (Novagen, Madison, WI, USA) harboring the recombinant plasmid was grown at 37 °C in LB medium (Luria-Bertani) containing ampicillin (100 µg/ml) until an OD600 = 0.8 was reached and the cells were induced to a final concentration of 1 mM isopropyl 1-thio-β-Dgalactopyranoside (IPTG). Cells were harvested 4 h after induction, resuspended in 4 times the pellet weight of lysis buffer (50 mM TRIS, 150 mM NaCl, 1 mM DTT, pH 7.5, ½ Protease inhibitor tablet [Complete, EDTA-free, Roche Diagnostics GmbH, Mannheim, Germany]), and lysed by using a micro-fluidizer (Microfluidics, Newton, MA, USA). After centrifugation Polyethylenimine (Fluka, Basel, Switzerland) was added drop-wise under continuous stirring to the supernatant to a final concentration of 0.6%. The lysate was incubated for 20 min and centrifuged at 12.000 g. Ammonium sulfate was added drop-wise to a final concentration of 60% under stirring to the supernatant. The lysate was centrifuged at 12.000 g and the pellet was solved in 30 ml of buffer A (50 mM TRIS, pH 7.5). The lysate was afterwards dialyzed against 2 x 4 l buffer A overnight. The lysate was applied to a HeparinFF column (GE Healthcare, Munich, Germany) using a step gradient with increasing NaCl concentrations (0 -1 M). For further purification the eluted fractions containing NusG were pooled and concentrated with Vivaspin concentrators (Vivascience, MWCO 5 kDa). The concentrated sample was applied to an S75 gel filtration column (50 mM TRIS, 150 mM NaCl;GE Healthcare, Munich, Germany). The fractions containing NusG were pooled and dialyzed against buffer as used for NMR measurements (NMR-buffer: 10 mM potassium phosphate, 50 mM NaCl, pH 6.4). The identity and structural integrity of purified protein was analyzed by SDS-PAGE as well as by NMR spectroscopy. NusG-NTD (1-124) was cloned via BPU1102I and NdeI into the E. coli expression vector pET11A (Novagen, Madison, WI, USA). The same procedure as described above for fulllength NusG was used for expression and purification of NusG-NTD. NusG-CTD (123-181) was cloned and purified as described before [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF].

Full-length NusG mutation S16C: For NusG S16C the following primers were used: 5'-primer: GTC GTT CAG GCG TTT TGC GGT TTT GAA GGC CGC, 3'-primer: GCG GCC TTC AAA ACC GCA AAA CGC CTG AAC GAC. The mutation was introduced by using the QuikChange protocol (Stratagene, La Jolla, CA, USA). Expression and purification were as for full-length NusG, except that 1 mM DTT was added to all buffers.

Random spin labeling of the ε-amino groups of lysines: Spin labeling of the lysines with 1-Oxyl-2,2,5,5-tetramethylpyrolline-3-carboxylate N-hydroxysuccimide Ester (OXYL-1-NHS; Toronto research Chemicals Inc., North York, ON, Canada) was done according to a slightly modified published protocol [START_REF] Lawrence | Spin-label study of histone H1-DNA interaction. comparative properties of the central part of the molecule and the N and C-amino tails[END_REF]. Briefly, 500 µM protein solution in NMR buffer (10 mM potassium phosphate, 50 mM NaCl, pH 6.4) were washed with 10 mM sodium carbonate buffer, pH 9.2, for buffer exchange in Vivaspin concentrators (Vivascience, MWCO 5 kDa). A 10 fold excess of OXYL-1-NHS in DMSO was added, followed by incubation for one hour at room temperature and an additional hour at 4 °C in the dark. To remove unreacted OXYL-1-NHS, the sample was washed with 20 ml of NMR-buffer in a Vivaspin concentrator. For reduction of the spin label, ascorbate (500 mM stock) was added directly to the NMR tube to a final concentration of 5 mM.

Site specific spin labeling of NusG cysteine mutant: Spin labeling of the cysteines introduced into

NusG S16C with (1-Oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl) Methanethiosulfonate (MTSL; Toronto research Chemicals Inc., North York, ON, Canada) was done according to a slightly modified published protocol [START_REF] Berliner | A novel reversible thiol-specific spin label: Papain active site labeling and inhibition[END_REF]. Briefly, 500 µM protein solution in NMR buffer was washed with 10 mM acetate buffer for buffer exchange in Vivaspin concentrators (Vivascience, MWCO 5 kDa). After addition of DTT to a final concentration of 5 mM, the sample was kept at 4 °C for one hour.

For removal of DTT, the solution was eluted isocratically with 10 mM acetate from a HiTrap Desalting column (GE Healthcare, Munich). A 10 fold excess of MTSL dissolved in acetonitrile was added, followed by incubation overnight at room temperature in the dark. To remove unreacted MTSL, the sample was again washed with 20 ml of NMR-buffer in a Vivaspin concentrator. For reduction of the spin label, ascorbate (500 mM stock) was added directly to the NMR tube to a final concentration of 5 mM. NMR: NMR experiments were recorded at sample temperatures of 298 K on Bruker Avance 600 MHz and 700 MHz (equipped with a cryogenically cooled probe) NMR spectrometers. Assignments for NusG-NTD and full-length NusG were from previous work [START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF]. The isolated NusG-CTD 1 H, 15 N heteronuclear single quantum coherence (HSQC) resonances matched the corresponding signals of full-length NusG perfectly and were trivially assigned and the assignments verified by triple resonance NMR experiments.

Paramagnetic relaxation enhancement (R2,para = R2,spinlabel -R2,no spinlabel) was determined by a two point scheme using an HSQC experiment with additional spin echo period during the first INEPT transfer [30]. Spin echo intervals were set to 0.1 ms and 10.2 ms. 15 N longitudinal (R1) and transverse (R2) relaxation rates were determined by standard methods at a 1 H frequency of 600.2 MHz and 700.2 MHz. Relaxation curves were fitted to monoexponential decays using the program package CURVEFIT (A.G. Palmer, Columbia University, USA). Rotational correlation times were determined from the R2/R1 ratios using the program TENSOR2 [START_REF] Dosset | Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data[END_REF].

Molecular Dynamics Simulation:

In order to test the possibility of an intramolecular inter-domain interaction of E. coli NusG analogous to the swapped dimer interaction in A. aeolicus NusG, we calculated the time-dependent structure with molecular dynamics simulations. An initial structural model of E. coli NusG with domain interaction was constructed by fitting the solution structures of the E. coli domains to A. aeolicus NusG. The simulation using XPLOR (XPLOR-NIH 2.1.2 [START_REF] Schwieters | The xplor-NIH NMR molecular structure determination package[END_REF]) was based on our standard protocol for protein structure calculation [START_REF] Schweimer | Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase lck[END_REF]. During the calculation, backbone coordinates were held fixed for all residues except the linker region between NTD and CTD (residues 117-126) and the sequence region 99-104 to reduce steric clashes between NTD in CTD in the initial structure. Additionally, residues in the flexible region E48-R62 were removed in silicio to avoid steric conflicts. No further restraints were applied. Existence of an intramolecular inter-domain interaction was accepted based on the absence of violation of restraints (deviation of bond lengths and angles), and 50 structures out of total 80 calculated were accepted based on this criterion.

Results and Discussion

NMR titrations suggest independence of the NusG domains: 1 H, 15 N-HSQC chemical shifts of resonances from isolated NusG-CTD and isolated NusG-NTD were virtually identical to the chemical shifts of the respective domains in full-length NusG (Fig. S1), rendering persistent domain interactions within full-length NusG highly unlikely. In order to further clarify whether or not transient domain interactions exist for the more general case of the isolated domains we resorted to observation of HSQC chemical shift perturbations of an isotope labeled domain, NusG-CTD or NusG-NTD, upon addition of the unlabeled potentially interacting domain, a method that is well established as a tool to study the interplay between molecules [START_REF] Zuiderweg | Mapping protein-protein interactions in solution by NMR spectroscopy[END_REF]. NMR spectroscopy, due to its inherent insensitivity and its resulting requirement for sample concentrations in the high micromolar range, is useful for detecting even weak (kd ~ μM-mM) interactions, and perturbations of amide group resonance shifts as detected by 1 H, 15 N HSQC are very sensitive even to subtle structural changes. As NusG-CTD F165 was claimed to strongly interact with a NusG-NTD hydrophobic cavity [START_REF] Knowlton | A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants[END_REF], significant chemical shift changes are expected on domain contact at least for F165 and residues in its vicinity, that is residues in the loop between strands β3 and β4. Titration of unlabeled NusG-NTD to 15 N labeled NusG-CTD to a two-fold excess and vice versa, however, did not result in observable chemical shift changes in the NMR experiments (Fig. S2). Thus, with the level of sensitivity provided by HSQC perturbation experiments, domain interaction could be detected neither within the full-length protein nor for the isolated domains under our experimental conditions. This observation strongly correlates with our earlier conclusion that the area around F165 is not involved in mutual interactions of NusG domains, but is rather a key residue in NusE:NusG complex formation [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF].

15 N relaxation reveals decoupled domain reorientation: In order to study the degree of motional independence of the NusG domains we determined their 15 N relaxation rates in the context of the fulllength protein [START_REF] Kay | Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease[END_REF]. In a two-domain protein, concerted tumbling of domains can be described by a single rotational diffusion tensor, whereas independent tumbling of domains requires description with different rotational diffusion tensors [START_REF] Fushman | Determining domain orientation in macromolecules by using spin-relaxation and residual dipolar coupling measurements[END_REF]. In an isotropic rotation model, differences in these tensors are directly reflected in differences in the average relaxation rates. 15 N transverse and longitudinal relaxation rates were determined at 14.1 T, with a sample concentration of 200 μM to reduce aggregation (Fig. 1A,1B). In the HSQC spectrum of the fulllength protein, virtually all residues of NusG-CTD were observed, the average longitudinal relaxation rate (R1) was 1.35±0.09 sec -1 , and the transversal relaxation rate (R2) was 15.4±1.9 sec -1 , corresponding to a rotational correlation time of τc=10.1 ns in an isotropic model. Although the NusG-NTD signals were considerably weaker, 67 non-overlapping signals were observed and the average longitudinal relaxation rate was 1.1±0.2 sec -1 , the transversal rate 19.0±4.6 sec -1 , corresponding to τc=13.1 ns in the isotropic model. Analysis of R1/R2 distributions offers an elegant method to detect interdomain motion on a time scale faster than the overall tumbling [START_REF] Horstmann | Domain motions of the mip protein from legionella pneumophila[END_REF]. The R1/R2 ratios form the basis for determination of the rotational diffusion tensor by NMR relaxation, and for compact globular proteins a uniform distribution of R1/R2 ratios is characteristic [START_REF] Kay | Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: Application to staphylococcal nuclease[END_REF]. The bimodal distribution of the R1/R2 ratios (Fig. 1C) that reflect the two domains demonstrates their different rotational reorientation behavior, arguing against a stable domain interaction on the nanosecond timescale. Although the difference in relaxation rates demonstrates independent movement of the two domains to a certain degree, τc of the NTD and CTD in full-length NusG are higher than τc of the isolated domains (13.1 ns vs. 8.9 ns and 10.1 ns vs. 4 ns; see Fig. S3 for relaxation rates of the individual domains). This increase of rotational correlation times of individual isolated domains up to values found in multidomain proteins without any domain interaction has been observed for several proteins [START_REF] Zhou | Phosphotransfer and CheY-binding domains of the histidine autokinase CheA are joined by a flexible linker[END_REF][START_REF] Nakaseko | Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains[END_REF][START_REF] Konrat | Structure of cysteine-and glycine-rich protein CRP2. backbone dynamics reveal motional freedom and independent spatial orientation of the lim domains[END_REF][START_REF] Beglova | Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor[END_REF]. The spatial exclusion due to the presence of a second domain connected by a linker is likely hampering an unrestricted rotation of a domain resulting in a longer correlation time [START_REF] Nakaseko | Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains[END_REF][START_REF] Beglova | Backbone dynamics of a module pair from the ligand-binding domain of the LDL receptor[END_REF]. The large increase of the rotational correlation times of both NusG domains indicates that motional decoupling of NusG-CTD and NusG-NTD via the 5 residue linker is imperfect, and a significant contribution of this effect in full length NusG to the relaxation mechanisms is expected. T he interdomain motion alters the overall shape of full length NusG and therefore affects the overall tumbling. As consequence, complete uncoupling of domain dynamics and overall rotation is unlikely, and this coupled motions render a more detailed analysis at least very difficult [START_REF] Nakaseko | Adjacent zinc-finger motifs in multiple zinc-finger peptides from SWI5 form structurally independent, flexibly linked domains[END_REF]. In intact NusG, V162 and I164 of NusG-CTD exhibit significantly enhanced R2 rates of 21.5 sec -1 as compared to the domain average of 15.4 sec -1 , a difference not found in isolated CTD. Most likely the enhanced transverse relaxation can be attributed to a chemical exchange contribution, and as these residues are located in the domain interface of the swapped-dimer crystal structure, this might be regarded as initial evidence of transient domain interaction in solution.

Only highly sensitive PRE experiments show signs of interaction between the isolated domains :

Fast exchange on the NMR timescale leads to observation of population averaged parameters, and states that are extremely weakly populated on time average often cannot be detected even by HSQC chemical shift perturbations or changes in relaxation rates. Paramagnetic interactions, however, provide a means to detect even weak and transient interactions between molecules. Paramagnetic centers dramatically increase relaxation rates of nearby nuclei and, as a consequence, presence of even very minor concentrations of paramagnetic labels in proximity to observed nuclei can enhance the relaxation rate of the latter to an observable degree [START_REF] Tang | Visualization of transient encounter complexes in protein-protein association[END_REF]. Thus, PRE observed in a non spinlabeled protein in the presence of a spin-labeled protein points to an at least transient proximity of both molecules. The experiment is straightforward as a non-spin-labeled protein, for example NusG-CTD, can be made easily detectable in HSQC spectra by 15 N enrichment, and introduction of paramagnetic centers into proteins, for example NusG-NTD, is possible by random labeling of mostly surface exposed lysine residues with OXYL-1-NHS [START_REF] Jahnke | Spin label enhanced NMR screening[END_REF]. In such an experiment, addition of OXYL-1-NHS-NusG-NTD to 15 N-NusG-CTD (Fig. S4) causes observable and specific PRE in the latter, and vice versa (Fig. 2). Mapping of the resonances with increased relaxation rates onto the three-dimensional structure of NusG-CTD shows very clearly the region around F165 and the region P140 to N145, close to F165 in the turn between β 1 and β 2 , to be the ones most seriously affected (Fig. 2).

The outcome of the inverse experiment, that is addition of OXYL-1-NHS-NusG-CTD to 15 N-NusG-NTD (Fig. S4), was less clear-cut. The hydrophobic patch around F65 was clearly affected, together with several residues in helix α 3 and residues close to F65 that form an apolar surface surrounded by polar residues. However, several isolated residues in helices α 1 and α 2 at the opposite side of the molecule as well as several residues of the »upper« loop regions facing away from the putative interaction site were affected (Fig. 2). Combined, the PRE results yield a picture in which a plug around F165 fits nicely into a socket around F65. Although this is a reasonable contact surface, and although these sites correspond well to those found in the swapped dimer crystal structure [START_REF] Knowlton | A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants[END_REF], our negative results with HSQC chemical shift mapping indicate the interaction to be rather weak and transient. Under the assumption of chemical shift changes in the range typically observed in this experimental setup (e.g. NusG-CTD:NusE [START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF], λN:AR1 [START_REF] Prasch | Interaction of the intrinsically unstructured phage lambda N protein with E. coli NusA[END_REF], LckSH3:Tip [START_REF] Schweimer | Structural investigation of the binding of a herpesviral protein to the SH3 domain of tyrosine kinase lck[END_REF]) no significant chemical shift changes point to less than 10 % bound species in the NMR samples, which correspondents to a Kd higher than 5 mM.

PREs of site-specific full-length NusG are concentration dependent: Chemically modifying cysteines with MTSL provides a means to observe the PRE induced by site-specific labels. Thus, in order to clarify whether or not the weak domain interaction observed by random labeling in solution is intraor intermolecular, we performed additional PRE experiments on a full-length NusG construct, containing MTSL-cysteine S16C. PRE originating from MTSL-Cys was detected in the unchanged CTD, clearly indicating an interaction between NusG-NTD and NusG-CTD, thus neither supporting nor ruling out the formation of a domain-swapped dimer or an intramolecular inter-domain interaction within one molecule. To further probe these domain interactions, we studied the concentration dependence of the PRE induced by MTSL-Cys16 on the CTD as intramolecular domain interactions would clearly be expected to result in a concentration-independent PRE due to unchanged internuclear distance distributions in a stable structure with interacting domains, whereas a concentration dependent PRE would demonstrate intermolecular domain interactions by reduction of the fraction of interacting molecules upon dilution and decrease of the observed ensemble averaged PRE. The PRE thus determined at different protein concentrations (Fig. 3) shows a direct dependence of the relaxation enhancement on sample concentration, thus clearly out-ruling an intramolecular interaction and deciding for the model of a transient domain-swapped dimer formation, which is extremely scarcely populated.

E. coli NusG NTD:CTD interaction analogous to that observed within the swapped dimer in A. aeolicus NusG, but within a single molecule, would only be possible if no sterical restraints would exist in the linker sequence. To test whether or not such steric and dynamic restraints exist we performed molecular dynamics simulations using a structural model with interacting domains to explore the conformational space of the linker region. Only a slight rearrangement of the loop prior to helix α3 of NusG-NTD would be necessary to remove all sterical clashes in the initial model based on the solution structure of E. coli NusG. In addition, all 50 analyzed lowest energy structures are without violation of bonds or angles and provide possible linker conformations that could allow domain interactions within one NusG molecule (Fig. 4). Therefore, an intramolecular domain interaction cannot be ruled out solely on the argument of the linker being too short. Nevertheless, the linker region must adopt a very extended conformation for the domains to be able to directly interact. This is expected to lead to a high degree of rigidity in the linker on the timescale of molecular reorientation due to the low number of possible conformational states that the linker can adopt in this extended state. On the other hand, the 15 N transversal relaxation rates of G119 (7.6 Hz) and D120 (6.4 Hz) are significantly lower than those for the other residues in the respective domains (Fig. 1B). The reduction of R2 is characteristic for enhancement of mobility on the sub-τc (sub nanosecond) timescale, demonstrating experimentally the flexibility of the linker region in contrast to the rigidity of the linker needed for direct interaction as shown by the MD simulation. This renders a domain interaction within one molecule highly unlikely.

Conclusions: Our NMR studies on domain interactions of E. coli NusG show these interactions to be not detectable via standard chemical shift mapping, and 15 N-relaxation measurements reveal movements of the two domains to be independent to a very high degree. Domain interactions could only be observed with the extremely sensitive technique of PRE measurements, and it needed a combination of this technique and site-directed paramagnetic labeling to show that these weak interactions were inter-rather than intramolecular. Thus, a low population of molecules seems to adopt a bimolecular conformation compatible with the proposed swapped dimer observed in X-ray crystallography. Intramolecular domain interaction, however, would be required for an autoinhibitory function and therefore biologically relevant form to reduce the population of the active open state. The mode of regulation found for the NusG paralogue RfaH [START_REF] Belogurov | Structural basis for converting a general transcription factor into an operon-specific virulence regulator[END_REF] can thus be ruled out to work for NusG, which may explain why NusG maintains its overall function without additional activation signals, while RfaH needs the ops-site to render the protein functional. Current opinion is that NusG and RfaH derived from the same ancestor [15], possibly a NusG protein that exists in closed conformation and that may be regulated by domain interactions. It may further be speculated that regulation of NusG via domain interactions is no longer necessary when RfaH is present. This hypothesis awaits further structural investigation, because in addition to the proposed swapped-dimer for A. aeolicus [START_REF] Knowlton | A spring-loaded state of NusG in its functional cycle is suggested by X-ray crystallography and supported by site-directed mutants[END_REF] and the non-interacting T. thermophilus [START_REF] Reay | Structural and sequence comparisons arising from the solution structure of the transcription elongation factor NusG from thermus thermophilus[END_REF] NusGs, detailed structural information on other thermophilic NusGs is not available yet.

Fig. 1 A

 1 Fig. 1 A) Longitudinal relaxation rates (R1) for full-length NusG. B) Transversal relaxation rates (R2) for full-length NusG. Residues 45 to 65 and 115 to 125 are highly flexible and located in strongly overlapping regions of the protein so that their relaxation rates could not be determined. C) The distribution of R1/R2 is bimodal for full-length NusG. NusG-CTD, black bars; NusG-NTD, gray bars.

Fig. 2 A

 2 Fig. 2 A) Surface representation of the individual E. coli NusG domains (NusG-NTD PDB-ID: 2K06;NusG-CTD PDB-ID: 2JVV;[START_REF] Mooney | Two structurally independent domains of E. coli NusG create regulatory plasticity via distinct interactions with RNA polymerase and regulators[END_REF]) with secondary structure elements. F65 and F165, blue sticks; amino acids with an R 2,para (H N )-effect >20 Hz in the titrations of the individual domains, red. B) R2,para (H N )-rates for each amino acid upon titration with the individual spin-labeled domains, gray; signals disappearing due to extensive line broadening, red; 20 Hz significance level, dotted line.

Fig. 3

 3 Fig.[START_REF] Hartzog | Evidence that Spt4, Spt5, and Spt6 control transcription elongation by RNA polymerase II in saccharomyces cerevisiae[END_REF] Effects of spin-labeled NusG S16C on the R2,para (H N )-rates of NusG-CTD in full-length NusG. Effects at a protein concentration of 150 µM on selected amino acids, dark gray; effects at a protein concentration of 50 µM, light gray.

Fig. 4

 4 Fig.[START_REF] Burmann | A NusE:NusG complex links transcription and translation[END_REF] Superposition of the 20 lowest energy structures of molecular dynamics simulation with intramolecular inter-domain interactions (NTD, green; CTD, red; linker region (residues Q117-T126 ), blue). The flexible region E48-R62 (indicated by the dashed line in the left picture) was removed in silicio to avoid steric conflicts.

Figure 1 :

 1 Figure 1:

Figure 2 : 13 Biochemical

 213 Figure 2:

Figure 3 :Fig. 4 :

 34 Figure 3:

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

Acknowledgement

We thank Ramona Heissmann for excellent technical assistance.

Funding

This project was supported by a grant to PR from the Deutsche Forschungsgemeinschaft DFG (Ro617/16-1)