Molecular Cell Ar ticle
Rachel A. Mooney ,1 Sarah E. Davis ,1,4 Jason M and Robert Landick1 ,4 , * 1Department of Biochemistry 2Department of Genetics 3Genome Center 4Department of Bacteriology University of Wisconsin , Madison , WI 53706 , USA * Correspondence : landick@bact.wisc.edu DOI 10.1016 / j.molcel .2008.12.021 
Regulator Trafficking on Bacterial
SUMMARY
The trafficking patterns of the bacterial regulators of transcript elongation s , r , NusA , and NusG on 70 genes in vivo and the explanation for promoterproximal peaks of RNA polymerase ( RNAP ) are unknown . 
Genome-wide , E. coli ChIP-chip revealed distinct association patterns of regulators as RNAP transcribes away from promoters ( r first , then NusA , then NusG ) . 
However , the interactions of elongating complexes with these regulators did not differ significantly among most transcription units . 
A modest variation of NusG signal among genes reflected increased NusG interaction as transcription prog-resses , rather than functional specialization of elongating complexes . 
Promoter-proximal RNAP peaks were offset from s peaks in the direction of tran-70 scription and co-occurred with NusA and r peaks , suggesting that the RNAP peaks reflected elongating , rather than initiating , complexes . 
However , inhibition of r did not increase RNAP levels within genes downstream from the RNAP peaks , suggesting the peaks are caused by a mechanism other than r-dependent attenuation . 
INTRODUCTION
Transcription of genes by RNAP is controlled by a multiplicity of regulators that modulate template DNA conformation , control initiation , or govern RNAP 's progress through transcription units ( TUs ) in response to internal and environmental signals . 
In bacteria and eukaryotes , transcription regulators can be divided into those acting during transcript initiation , elongation , or termination . 
Precisely where initiation regulators release and elongation regulators associate with RNAP is unknown . 
Further , the distinction between these classes of regulators is not absolute ; some may act during multiple stages of transcription , possibly with different effects . 
Finally , although some elongation regulators are known to target subsets of TUs , it is unclear whether general elongation regulators like NusA , NusG , and r interact with most elongating complexes ( ECs ) equivalently or instead preferentially interact with certain TUs or sites within TUs . 
In bacteria , s initiation factors bind tightly to core RNAP ( consisting of b0 , b , a2 , and u subunits ) and determine the sequence specificity of RNAP-promoter interactions ( Figure 1A ) . 
ss are thought to be released shortly after RNA synthesis begins . 
However , whether s release occurs obligately or stochastically , whether s may be completely retained on a subset of TUs , and whether s may transiently rebind to the EC during elongation with possible regulatory consequence all remain in debate ( Bar-Nahum and Nudler , 2001 ; Kapanidis et al. , 2005 ; Mooney et al. , 2005 ; Mooney and Landick , 2003 ; Mukhopadhyay et al. , 
2001 ; Raffaelle et al. , 2005 ; Reppas et al. , 2006 ; Wade and Struhl , 2004 , 2008 ) . 
During or after promoter escape , the EC can associate with one or more elongation regulator ( Figure 1A ) . 
In bacteria , NusA and NusG alter EC properties differently via direct and independent interactions with RNAP and are the best characterized regulators of elongation ( Burns et al. , 1998 ; Greenblatt et al. , 1981 ; Li et al. , 1992 ; Linn and Greenblatt , 1992 ; Sullivan and Got-tesman , 1992 ) . 
NusA preferentially enhances transcriptional pausing associated with nascent RNA hairpins ( Artsimovitch and Landick , 2000 ; Farnham et al. , 1982 ; Greenblatt et al. , 1981 ; Yakhnin and Babitzke , 2002 ) , enhances intrinsic termination at some sites more than others ( Kassavetis and Chamberlin , 1981 ; Linn and Greenblatt , 1992 ; Yakhnin and Babitzke , 2002 ) , modulates r-dependent termination ( Burns et al. , 1998 ) , and is an essential component of antitermination complexes that form on ribosomal RNA ( rrn ) and phage l operons ( Mason et al. , 1992 ; Shankar et al. , 2007 ; Torres et al. , 2001 ) . 
NusG increases the rate of RNA chain extension , at least partly by decreasing pausing associated with backtracking ( Artsimovitch and Land-ick , 2000 ) , enhances r-dependent termination via interactions with RNAP and r ( Li et al. , 1992 , 1993 ; Sullivan and Gottesman , 
1992 ) , and also is a component of both rrn and l antitermination complexes ( Mason et al. , 1992 ; Torres et al. , 2001 ) . 
Despite these multiple roles of NusA and NusG , it is unclear whether they associate equivalently with ECs on all TUs , differentially with subsets of TUs , or differentially at locations within TUs . 
The homohexameric r protein terminates transcription after binding to unstructured , C-rich nascent RNA . 
RNA stimulation of its ATP-dependent translocase activity allows r to travel 50 to 30 along the RNA and dissociate ECs unless blocked by intervening ribosomes ( Richardson , 2002 ) . 
It is uncertain where within TUs r interacts with ECs and whether r preferentially affects a subset of TUs . 
The report of Reppas et al. ( 2006 ) that a significant fraction of TUs in E. coli exhibit promoter-proximal peaks of RNAP heightens interest in knowing whether promoter-proximal , r-dependent termination could contribute to the apparent decrease in RNAP density downstream from promoters . 
To investigate trafficking of these regulators on bacterial TUs and the reported promoter-proximal block to transcriptio 
( Reppas et al. , 2006 ) , we used `` chromatin immunoprecipitation '' ( Kuo and Allis , 1999 ; Solomon et al. , 1988 ) followed by microarray hybridization ( ChIP-chip ; Wade et al. , 2007 ) . 
Our study provides comparative analysis with improved resolution of some proteins examined previously ( RNAP , s , and NusA ; 70 Grainger et al. , 2005 ; Herring et al. , 2005 ; Raffaelle et al. , 2005 ; 
Reppas et al. , 2006 ; Wade and Struhl , 2004 ) , as well as genome-wide views of NusA , NusG , and r , leading to important insights into trafficking of bacterial transcription regulators . 
RESULTS
Analysis of RNAP ChIP-chip Signals on E. coli TUs
We applied ChIP-chip to E. coli K-12 at mid-log phase of growth at 37 C in defined minimal glucose medium ( Experimental Procedures ) , conditions in which many biosynthetic genes must be expressed and that were used previously for expression analysis ( Allen et al. , 2003 ) . 
Using specific antibodies targeting core RNAP , s , NusA , r , or a hemagglutinin ( HA ) epitope present in 70 three copies at the N terminus of the chromosomal nusG gene , we obtained associated DNA that was then fluorescently labeled and hybridized to a tiled oligonucleotide microarray ( 25 bp spacing ; Experimental Procedures ) . 
Initial analysis of the immu-noprecipitated DNAs relative to input DNA revealed excellent correspondence among the sites of enrichment by anti-s and 70 anti-RNAP ( anti-b0 ) antibodies ( Figure 1B ) . 
Closer examination ( e.g. , of the expanded region around 0.94 mb shown in Figure 1B ) revealed that s was predominantly associated with 70 DNA near promoters , whereas RNAP could be detected in association with both promoter and transcribed-region DNA . 
The strongest signals were in genes encoding tRNA , rRNA , and ribosomal proteins ( e.g. , serW and rpsA ) , as expected and reported previously ( Grainger et al. , 2005 ; Raffaelle et al. , 2005 ; Reppas et al. , 2006 ; Wade and Struhl , 2004 ) . 
NusA , NusG , and r were associated with ECs in most locations where RNAP was present . 
RNAP is known to associate nonspecifically with chromosomal DNA ( deHaseth et al. , 1978 ; Grigorova et al. , 2006 ; von Hippel et al. , 1974 ) . 
To estimate the corresponding nonspecific ( background ) ChIP-chip signal for RNAP , we examined regions of the bacterial chromosome thought to be devoid of transcription , such as the cryptic bglB gene ( Defez and De Felice , 
1981 ) . 
We identified 170 regions greater than 1 kb whose average RNAP ChIP signal was indistinguishable from that on bglB ( bkgd , Figure 1B ; gray box near 0.94 mB in expanded region ; Table S2 ) . 
The signals for these regions were normally distributed with a mean below the signal for 84 % of the complete genome-wide probe set ( compare black to blue histo-grams , Figure 1C ; Supplemental Experimental Procedures ) . 
This suggests that most of the E. coli genome is transcribed at levels above the nonspecific background , consistent with previous estimates ( Selinger et al. , 2000 ) . 
To characterize RNAP and regulator occupancy further , we identified `` high-quality '' TUs that were significantly above this background and for which signals from adjacent TUs did not obscure the pattern of RNAP and regulator association and dissociation ( e.g. , serS in the expanded region as opposed to clpA and cydCD , which were obscured by strong signals from the adjacent serW tRNA gene ) . 
We identified 109 such TUs , which were spread across the E. coli genome and represented a range of expression levels and TU lengths ( Figure 1D and Table S1 ) . 
Regulator Trafficking on Representative E. coli TUs
To gauge the basic patterns of regulator trafficking on these 109 TUs , we wished to scale the data in proportion to occupancy of regulators on DNA . 
Although true occupancy is impossible to measure without knowing the relative efficiencies of crosslinking for each protein at each TU location as well as the signals corresponding to zero and full occupancy , we nevertheless defined an apparent occupancy ( Occapp ) by linearly scaling signals for each protein between zero , which was set equal to the background defined by bglB-similar regions ( Figure 1C ; Table S2 ) , and one , which was arbitrarily defined as the average of the ten threeprobe clusters with highest average value ( Figure 1C ; Supplemental Experimental Procedures ) . 
Therefore , Occapp is a function of true occupancy and relative `` crosslinkability . '' 
An examination of eight representative TUs ( seven from among the 109 high-quality TUs plus rrnE ) revealed significant variation both in the uniformity of RNAP and regulator Occapp across TUs and in the ratios of RNAP Occapp to s and other 70 regulators at locations within TUs ( Figure 2 ) . 
In some cases , the peak of s Occapp surrounding the transcription start site 70 ( TSS ) was much greater than RNAP Occapp , with the latter exhib-iting a relatively uniform distribution across the TU ( serS , rspF , and acnB ; Figures 2A , 2D , and 2F ) . 
In other cases , the s 70 peak was more similar to the corresponding RNAP Occapp ( atpI BEFHAGDC , gltBDF , and carAB ; Figures 2C , 2E , and 2H ) ; in these cases RNAP typically exhibited a pronounced promoterproximal peak similar to that previously reported ( Reppas et al. , 2006 ; Wade and Struhl , 2008 ) . 
These representative examples suggest there is no one-to-one correspondence between s Occapp and RNAP Occapp at promoters ; this obser-70 vation was reflected in the modest ( 0.77 ) correlation between peak Occapp values for s and RNAP ( Figure S1 ) . 
70 
Patterns The regulators s , NusA , NusG , and r all appeared to be 70 present on each TU , but with notable differences in their Occapp distributions . 
NusA closely mirrored RNAP on each TU , appearing to associate with RNAP as the signal from s disappears . 
70 This is consistent with the long-standing view that NusA displaces s during transcript elongation ( Greenblatt and Li , 70 1981 ) . 
In contrast , NusG appeared to associate with elongating 
RNAP farther from promoters and did not appear to be present at locations where RNAP forms promoter-proximal peaks . 
Rather , NusG Occapp rose gradually to levels that exceed other regulators on most TUs . 
The ratio of NusG/RNAP Occapp appeared to be much greater in the distal portions of some TUs ( e.g. , atpIBEFHAGDC and cyoABCDE ; Figures 2C and 2G ) than others ( e.g. , rrnE and rpsF-priB-rpsR-rplI ; Figures 2B and 2D ) . 
The different pattern of NusG on rrnE may reflect its participation ( with NusA , NusB , NusE , and a subset of ribosomal proteins ) in the rrn antitermination complex ( Torres et al. , 2001 ) . 
r exhibited a striking pattern of significant promoter-proximal peaks near s peaks and RNAP promoter-proximal peaks , 70 but a lower Occapp over most of the TU . 
Finally , although s 70 
Occapp for the rrnE TU and seven representative TUs from among the 109 TUs selected for the absence of interfering upstream or downstream signals ( Figure 1D and Table S1 ) . 
Occapp was calculated as described in the Supplemental Experimental Procedures using two rounds of sliding-window smoothing ( 500 bp window for RNAP , NusA , NusG , and r ; 175 bp window for s ) . 
Genes are depicted as labeled open arrows , promoters as vertical lines 70 capped with arrows , and known intrinsic terminators as hairpins . 
Note that the scales of Occapp and TU length ( in kb , denoted by hatchmarks ) differ in each panel . 
Protein-encoding genes are colored blue , and the rRNA TU is colored yellow . 
Regulators are colored as in Figure 1 . 
Vertical dotted lines are the center of the s peak . 
For the rrn TU , there are two promoters ( and two s peaks ) . 
70 70 ( A ) serS , a monocistronic TU encoding seryl-tRNA synthetase . 
( B ) rrnE , one of seven E. coli rRNA TUs . 
Due to near-sequence-identity among the rRNA TUs , these signals represent the average of all seven rRNA TUs . 
( C ) atpIBEFHAGDC , the nine-gene TU encoding the F0 , F1 ATP synthase . 
( D ) rpsFpriBrpsRrplI , encoding the ribosomal protein S6 , DNA replication primosome protein N , ribosomal protein S18 , and ribosomal protein L9 . 
( E ) gltBDF , encoding glutamate synthase large and small subunits and a peri-plasmic protein involved in nitrogen metabolism . 
( G ) cyoABCDE , encoding cytochrome bo terminal oxidase and heme O synthase . 
( H ) carAB , encoding carbamoyl phosphate synthetase . 
was principally present at promoters , as reported previously ( Re-ppas et al. , 2006 ; Wade and Struhl , 2004 ) , s Occapp remained 70 above zero across most TUs ( e.g. , serS , rrnE , and cyoABCDE ; Figures 2A , 2B , and 2G ) . 
To examine the correlation between RNAP and regulator presence on TUs more carefully , we calculated the average ChIP ¬ 
( A ) Diagram illustrating calculation of mid-TU signals . 
For each of the 109 highquality TUs , the log2 ( IP/input ) signals for all probes within a 200 bp window surrounding the center of the TU were averaged to yield an estimate signal due to elongating RNAP or regulator associated with the elongating RNAP . 
Regulators are colored according to Figures 1 and 2 . 
( B ) Correlation of s and RNAP mid-TU signals . 
Only TUs for which the mid-70 TU point was more than 500 bp from the s peak were included ( to avoid influ-70 ence of signal from the s peak ; n = 80 ) ; r = 0.68 ; p < 0.001 . 
70 ( C ) Correlation of NusA and RNAP mid-TU signals ( n = 109 ) ; r = 0.97 ; p < 0.001 . 
( D ) Correlation of NusG and RNAP mid-TU signals ( n = 109 ) ; r = 0.71 ; p < 0.001 . 
( E ) Correlation of r and RNAP mid-TU signals ( n = 109 ) ; r = 0.78 ; p = < 0.001 . 
( F ) The correlation coefficient between the RNAP signal and each of the regulator signals plotted versus mean mid-TU signal for the regulator . 
Mean signals for NusA , NusG , s , and r are 73 % , 95 % , 33 % , and 51 % of mean RNAP 70 signals , respectively . 
chip signals for each in a 200 bp window in the middle of the 109 high-quality TUs ( Figure 3A ) and compared the regulator and RNAP ChIP-chip signals directly ( Figures 3B -- 3F ) . 
Strikingly , s , NusA , NusG , and r mid-TU signals all exhibit an obvious 70 correlation with RNAP mid-TU signals . 
However , the correlation was much greater for NusA than for s , NusG , or r ( Figure 3F ) . 
70 For s and r , the weaker correlation is consistent with lower 70 signal-to-noise ratio resulting from the reduced mean signals in the middle of the TUs . 
However , this is not the case for NusG , where the mean mid-gene signal was as large as the RNAP signal despite the much-reduced correlation ( Figure 3F ) . 
These results suggest that elongating RNAPs do not exhibit TU-specific variations in affinity for s , NusA , NusG , or r. Although the rela-70 tive affinity of each regulator for ECs differs ( i.e. , s and r exhibit 70 lower signals than NusA and NusG ) , there is no indication that they target one subset of TUs relative to others . 
Thus , they can rightly be classified as general elongation regulators as opposed to specialized regulators like RfaH that are recruited to a specific subset of TUs ( Artsimovitch and Landick , 2002 ) . 
To resolve the pattern of s , NusA , NusG , and r interactions 70 with RNAP more accurately , we took advantage of the similarity of these interactions among TUs to compute aggregate Occapp profiles ( Figure 4 ) . 
For this purpose , we selected a set of highly transcribed TUs among the 109 high-quality TUs ( to improve signal-to-noise ratios ) and avoided TUs known to contain transcription attenuators ( e.g. , trp or leu ) or multiple promoters that might complicate the distribution of RNAP . 
This yielded a set of 42 TUs that included 13 lacking an obvious promoter-proximal RNAP peak and 29 containing a readily discerned promoterproximal RNAP peak ( traces B and C in Figure 4A ) . 
We computed the aggregate Occapp for these TUs by aligning them relative to the genome coordinate of their s peak and then averaging 70 normalized Occapp values for each protein ( normalized relative to the highest Occapp for that protein in a given TU ) . 
The RNAP peak aggregate Occapp for the 42 TUs was offset in the direction of transcription from the s peak by 150 bp ( d in Figure 4A ) . 
The 70 size of this offset was widely distributed among different TUs and was uncorrelated with RNAP mid-TU signal ( Figure S2 ) . 
However , the 29 TUs exhibiting pronounced peaks were , on average , longer ( 3.43 kb average length ) , whereas the TUs on which Occapp declined much more slowly were , on average , shorter TUs ( 1.36 kb average length ; Mann-Whitney p < 0.001 ) . 
The aggregate Occapp profiles highlighted differences in regulator trafficking on E. coli TUs . 
s appeared to dissociate from 70 
RNAP as RNAP loses contact with the promoter ( as reported previously by Raffaelle et al. , 2005 ; Reppas et al. , 2006 ; Wade and Struhl , 2004 ) . 
Although the s peak was nearly symmetric 70 around its center as noted by Reppas et al. ( 2006 ) , it was skewed 20 bp downstream at its vertical midpoint in our data ( Figure S3 ) . 
This s skew was caused by translocation of 70 
RNAP relative to the TSS , as evidenced by loss of the skew and a slight upstream shift of the s peak upon treatment of 70 cells with rifampicin ( Figure S3 ) . 
Conversely , NusA appeared to associate fully with elongating RNAP sometime after the s 70 signal disappeared ( Figures 4B and 4C ) . 
Both the NusA and r aggregate profiles exhibited promoter-proximal peaks , as observed for the individual profiles ( compare Figures 2 and 4C ) . 
However , the r peak was displaced 50 bp upstream ( relative to the RNAP peak ) , whereas the NusA peak was displaced downstream . 
Finally , NusG associated with elongating RNAP much more slowly than either NusA or r , reaching a plateau of Occapp 800 bp downstream of the s peak . 
The same aggre-70 gate and individual-TU patterns of NusG association were observed using anti-NusG polyclonal antibody ( Figure S4 ) , ruling 
Taken together , our analysis of regulator trafficking on E. coli TUs ( Figures 2 -- 4 ) leads to the following key conclusions . 
First , s crosslinks almost exclusively to promoter DNA , although 70 a downstream skew of the s peak and weak s signal in the 70 70 middle of TUs are consistent with stochastic release of s 70 from elongating RNAP followed by weak s association with 70 ECs ( Mooney et al. , 2005 ) . 
The extent of s - EC association is 70 difficult to assess from ChIP-chip data ( see Discussion ) ; we can not exclude the possibility that nonspecific antibody-EC interaction contributes to the mid-TU s . 
70 Second , NusG associates with ECs more slowly than NusA on most TUs ( Figures 2 and 4 ) , except on antiterminated rrn TUs , where its faster association likely reflects incorporation into an antiterminated EC ( Torres et al. , 2001 ) . 
Conversely , the slower association of NusG on other TUs may suggest that its binding is stimulated by a feature of the EC that increases the farther RNAP transcribes . 
Third , r is evident at most TU locations , with a peak interaction at locations in between the strongest s and RNAP signals 70 ( Figures 4B and 4C ) . 
This suggests that r may associate with transcripts shortly after the initiation of transcription . 
r is detect-able throughout TUs , and the extent of this interaction is well correlated with the amount of RNAP located on the TU ( Figure 3E ) . 
This is consistent with the generally accepted role of r in premature termination whenever translation is compromised . 
To investigate the greater variability of NusG/RNAP ratios and NusG 's apparently slower association with ECs , we computed the NusG/RNAP , NusA/RNAP , and r/RNAP ratios for each gene and examined these ratios as a function of the average RNAP signal per gene ( Figures 5A -- 5C ) . 
NusA and r both exhibited relatively uniform distributions ; genes with low RNAP signals exhibited higher ratios ( as expected mathematically ; Figures 5A and 5B ) . 
In this analysis , NusA/RNAP ratios on rRNA genes were slightly above the trend line but were still consistent with at least 1:1 NusA : RNAP on most ECs . 
tRNA genes exhibited disproportionately high ratios of both NusA and r , suggesting that transcription of tRNA genes may differ from protein-coding genes . 
Small RNA ( sRNA ) genes , in contrast , exhibited normal ratios of NusA and r to RNAP . 
The NusG/RNAP ratio distribution differed strikingly from the 
NusA or r ratios . 
Although rRNA genes exhibited high NusG / RNAP ratios , a subset of genes with lower average RNAP signal exhibited even higher NusG/RNAP ratios ( inset , Figure 5C ) . 
Interestingly , several of these were genes involved in energy production ( genes from the nuo and cyo operons ) , murein/peptidoglycan biosynthesis and recycling ( oppD & F , murB & E ) , or amino-acid biosynthesis ( trpA & B , metI , cysM ) . 
This raised the possibility of a functional connection to elevated NusG levels on certain TUs ( e.g. , to localize transcription of certain genes ) . 
As an alternative , we considered whether the length of TUs might explain the abnormal NusG/RNAP ratios ( e.g. , if long TUs acquire higher NusG occupancy ) . 
To test this , we compared the NusG/RNAP ratio to the distance of genes from their TSS ( for cases where the TSS is known ) and found a strong correlation of TSS-gene distance to NusG/RNAP ratio ( Spearman r = 0.57 ; Figure 5D ) . 
Genes that deviated significantly from this strong correlation by exhibiting low NusG/RNAP ratios included rfa and rfb genes ( inset , Figure 5D ) . 
This is readily explained because rfa and rfb genes are regulated by RfaH , a specialized paralog of NusG that competes with NusG for interaction with ECs ( Belogurov et al. , 2007 ) . 
We conclude that the gradual increase in NusG association as transcription progresses , rather than a connection to gene function , explains elevated NusG/RNAP ratios on some genes . 
The high NusG/RNAP ratios on energy-related and amino-acid-biosynthetic operons simply reflect the greater-than-average length of these TUs . 
To confirm this interpretation , we plotted the average NusG/RNAP ratios for different gene functional classes by the average TSS-gene distance for the functional class ( Figure 5E ) . 
Classes with NusG/RNAP signal ratios below the genome average ( red circle , Figure 5E ) contained , on average , shorter genes , whereas classes exhibiting significantly higher NusG/RNAP signal ratios contained longer genes . 
Thus , the primary determinant of NusG levels is TSS-gene distance , rather than gene function . 
Promoter-Proximal RNAP Peaks Correlate with Promoter-Proximal NusA and r Peaks
Promoter-proximal RNAP peaks have been detected in E. coli and Drosophila and are suggested to reflect RNAPs kinetically blocked early in elongation ( for Drosophila ) or possibly even prior to promoter escape ( for E. coli ; Core and Lis , 2008 ; Muse et al. , 
2007 ; Reppas et al. , 2006 ; Wade and Struhl , 2008 ; Zeitlinger et al. , 2007 ) . 
Therefore , we asked whether promoter-proximal RNAP peaks were associated with NusA and r , which presumably requires promoter escape . 
We first calculated the traveling ratio ( TR ; the ratio of RNAP signal in the promoter-proximal peak to that within the TU ; Reppas et al. , 2006 ) for a set of genes with a 50-s70 peak and that were greater than 1 kb in length ( to insure the peak and mid-gene signals were well separated ; Figure 6A ) . 
We then tested whether a NusA peak , r peak , or both occurred within 300 bp of the RNAP peak and binned the results based on TR ( Figure 6B ) . 
If the RNAP peaks reflect RNAPs poised prior to promoter escape , then the fraction of RNAP peaks with NusA or r copeaks should decrease at low TR ( because a low TR would indicate promoter-bound RNAP that should not recruit NusA or r , in contrast to ECs that can bind both ) . 
Instead , we observed little change in the frequency of NusA and r copeaks at low TR . 
We also binned the frequency of NusA and r copeaks based on gene expression level ( Allen et al. , 2003 ) , to ask if a block to promoter escape correlates with low expression ( as suggested previously by Reppas et al. , 2006 ; Figure 6C ) . 
No correlation was evident . 
Further , the frequency of copeaks correlated to RNAP peak height ( Figure 6D ) , suggesting that the failure to detect NusA or r copeaks for a fraction of RNAP peaks 
( 25 % ) is mostly explained by false negatives in the peak-calling algorithm , since the signal-to-noise ratio for RNAP is better than that for NusA or r. Taken together , these results suggest that promoter-proximal RNAP peaks reflect RNAPs that have escaped promoters , at which point signals for NusA and r become detectable . 
To verify that RNAP peaks reflected premature termination rather than a block to promoter escape , we used quantitative RT-PCR to test representative sets of TUs that exhibited or lacked RNAP peaks ( Figures 4B and 4C ) for a drop in RNA transcript levels . 
This is an imperfect test because RNAs generated by premature termination are more difficult than long mRNAs to quantify accurately and also may be unstable . 
Nonetheless , six of eight TUs exhibiting RNAP peaks produced significantly more RNA near the 50 end versus 0 of 4 for TUs lacking RNA peaks ( Figure S5 ; p < 0.005 ; Student 's t test ) . 
Thus , most RNAP peaks are associated with premature transcription termination . 
Reppas et al. ( 2006 ) raised the possibility that RNAP peaks might instead correspond to RNAPs poised prior to promoter escape in part because they found 300 s peaks 70 not associated with detectable mRNAs . 
Thus , we asked if these s peaks exhibited NusA or r copeaks . 
Of the 300 70 peaks , 20 correspond to highly expressed stable RNA genes ; 138 of the remainder were associated with an RNAP peak ( Table S6 ) . 
Of these 138 , 74 were within 300 bp of s and 70 RNAP peaks in our data . 
Of these 74 , 45 ( 61 % ) were associated with a NusA peak ; 49 ( 66 % ) were associated with a r peak ; 33 ( 46 % ) were associated with both ; and 13 ( 18 % ) were associated with neither ( Figure S6 ) . 
As noted above , some NusA and r copeaks for small RNAP peaks were probably missed . 
Nonetheless , a few RNAP peaks likely represent promoter-bound enzyme : of three examples specifically cited by Reppas et al. ( 2006 ) , one ( hepA ) was associated with NusA and r , but two ( deoB and yjiT ) were associated with neither ( data not shown ) . 
The finding that promoter-proximal RNAP peaks correspond to RNAPs blocked early in elongation raised the possibility they result from transcriptional attenuation . 
Indeed , the Occapp profiles of genes regulated by attenuation resembled the aggreproximal peaks , could cause the RNAP peaks by r-dependent attenuation before a ribosome can bind and initiate translation , we examined the effect of the well-characterized r inhibitor , bicyclomycin ( Supplemental Experimental Procedures ) . 
If the RNAP peaks were caused by r-dependent attenuation , they should be reduced when cells are treated with bicyclomycin . 
genes that exhibit low TRs ( Cardinale et al. , 2008 ; Figure S8 ) . 
Thus , r-dependent attenuation does not appear to be the principal cause of promoter-proximal RNAP peaks . 
DISCUSSION
Our ChIP-chip study of the distributions of RNAP , s , NusA , 70 NusG , and r on E. coli TUs reveals the patterns of trafficking for regulators most central to control of transcript elongation in bacteria and has important implications for understanding the mechanisms underlying these patterns . 
s , NusA , NusG , and 70 r are distributed relatively uniformly among most transcribing RNAP molecules with apparent relative affinities for elongating RNAP of NusAzNusG > r > s70 . 
As RNAP moves away from a promoter , crosslinking of s greatly decreases . 
r and NusA 70 appear to associate with RNAP as s association decreases , 70 with r slightly preceding NusA , whereas NusG associates with elongating RNAP more slowly . 
As previously reported ( Reppas et al. , 2006 ) , RNAP exhibits strong promoter-proximal peaks on many , but not all TUs . 
We find that these peaks correspond to ECs and that they do not result from r-dependent attenuation . 
NusA, NusG, and r Exhibit Different Patterns of EC Association, but No TU-Specific Specialization
Our finding that NusA , NusG , and r are , to a first approximation , uniformly associated with ECs on most TUs suggests they act as general modulators of transcript elongation with about equal probability of altering responses of RNAP to intrinsic pause , arrest , or termination sites , regardless of where these sites occur in the genome . 
Due to the limited resolution of ChIP-chip , this does n't preclude specific associations of regulators at intrinsic sites that affect only a minority of elongating RNAP molecules or at which events occur rapidly relative to movement of RNAP over the surrounding DNA sequences . 
The results do rule out the possibilities that NusA , NusG , or r associate with certain TUs or certain sites within TUs to the exclusion of other TUs or locations . 
Nonetheless , each regulator associates with ECs as they move away from promoters in a distinct , regulator-specific pattern that is similar on most TUs ( Figure 7 ) . 
NusA exhibits negligible signal at promoters and associates with RNAP as s association is lost , closely paralleling RNAP 70 levels once RNAP moves away from a promoter ( Figures 2 -- 4 ) . 
NusA 's highest affinity contacts occur between the NusA CTD and the a-subunit CTD ; additional contacts are made by NusA 's KH and S1 domains to the nascent RNA and by the NusA NTD to a second site on RNAP , which may include the b-subunit flap tip ( Liu et al. , 1996 ; Mah et al. , 2000 ; Toulokhonov et al. , 2001 ) . 
At promoters , the a CTD binds to upstream DNA , either sequence specifically at UP elements or nonspecifically in association with s ( Estrem et al. , 1999 ) , and s region 4 occupies the 70 70 flap tip until nascent RNA reaches 16 -- 17 nt in length ( Murakami et al. , 2002 ; Nickels et al. , 2006 ) . 
Thus , NusA contacts are either not possible ( to nascent RNA ) or masked by DNA or s until 70 RNAP moves away from the promoter , at which point the association of NusA with the a CTD and nascent transcript likely tether NusA to the EC via interactions that are largely independent of EC position in a TU ( Figure 7 ) . 
Like NusA , NusG exhibits negligible signal at promoters , but unlike NusA , it appears to associate with RNAP in two phases . 
In the first phase , evident in aggregate Occapp profiles ( Figure 4 ) , NusG increases association with RNAP rapidly to 1 kb downstream from promoters . 
This first phase is distinct signal does not mirror the promoter-proximal RNAP peaks ( Figure 4C ) . 
In the second phase , NusG Occapp increases more slowly , resulting in the increased NusG/RNAP ratios for genes farther from promoters ( Figures 5D and 5E ) . 
One explanation for the delayed association pattern of NusG could be competition with s for its binding location on RNAP . 
70 
NusG is suggested to bind RNAP via contacts to the clamp helices ( Belogurov et al. , 2007 ) , which also make the tightest RNAP contact to s ( via s region 2 ; Arthur and Burgess , 70 70 1998 ; Young et al. , 2001 ) . 
Although s region 4 dissociates 70 from the flap tip when 16 -- 17 nt of RNA are synthesized , the s region two-clamp helices interaction can persist in the EC 70 without steric conflict ( Mooney et al. , 2005 ) . 
In this case , slow NusG association could reflect delayed dissociation of s 70 region 2 . 
This would mean that s dissociates from RNAP 70 more slowly than reported by the ChIP-chip assay , which instead shows a sharp fall-off in s crosslinking immediately down-70 stream from promoters ( Figure 4 ; Raffaelle et al. , 2005 ; Reppas et al. , 2006 ; Wade and Struhl , 2004 ; see below ) . 
Alternatively , s may release rapidly and NusG binding could require long 70 RNA transcripts , since it has been suggested that NusG contains an RNA-binding activity ( Steiner et al. , 2002 ) . 
r associates with TUs closer to promoters than either NusA or NusG and then appears to decrease somewhat in TU association farther from promoters , with an approximately uniform association relative to RNAP signal ( Figures 3 and 4 ) . 
The location of the promoter-proximal r peak is consistent with the requirement of 80 -- 100 nt for r effects on ECs ( Lau and Roberts , 1985 ) . 
Thus , r appears to bind as soon as the requisite nascent transcript becomes available but perhaps fails to terminate transcription because NusG is not yet associated with RNAP . 
This early binding could position r to detect and subsequently terminate synthesis of the occasional mRNA on which translation fails . 
The strong r ChIP signal may be reduced once ribosomes load 
Our analysis of s confirmed prior reports that the great majority 70 of s ChIP signal is lost as RNAP escapes the promoter ( Raf-70 faelle et al. , 2005 ; Reppas et al. , 2006 ; Wade and Struhl , 2004 ) , but asymmetry of the s aggregate Occapp peak suggests the 70 signal is lost on average 20 bp into TUs ( Figure S3 ) . 
However , a low s ChIP signal was present and was correlated with RNAP 70 signal at the middle of TUs ( Figure 3B ) . 
This likely reflects s - EC 70 interaction , although we can not exclude other possibilities ( e.g. , that transcription increases nonspecific binding of s - contain-70 ing holoenzyme to DNA , for instance by removing nucleoid proteins from DNA ) . 
In any case , it is difficult to assess the extent of the interaction from the low s ChIP signal because it may 70 reflect far less efficient s crosslinking to DNA than for promoter 70 complexes ( e.g. , indirect s - RNAP and RNAP-DNA crosslinking 70 in ECs rather than direct s - promoter DNA crosslinking ) . 
Our 70 results are consistent with the view that s breaks DNA contact 70 when RNAP escapes a promoter after which s 's weakened 70 contacts to RNAP allow its stochastic release ( Mooney et al. , 2005 ; Raffaelle et al. , 2005 ; Shimamoto et al. , 1986 ) but still support at least a weak equilibrium association with ECs and s rebinding at promoter-like sequences encountered during 70 
The Mechanistic Basis of Promoter-Proximal RNAP Peaks
In principle , promoter-proximal RNAP peaks could reflect one of at least three mechanistically distinct types of blocks to transcription . 
RNAP could be trapped ( 1 ) prior to promoter escape ( e.g. , before strand opening or in abortive initiation ) ; ( 2 ) early in elongation in a paused ( or poised ) state from which it can be released to productive elongation ; or ( 3 ) by premature and presumably regulated transcription termination ( transcriptional attenuation ) . 
Promoter-proximal RNAP peaks are common for human and Drosophila genes where they appear to be correlated with developmentally regulated rather than with housekeeping genes ( ENCODE Project Consortium , 2004 ; Guenther et al. , 2007 ; Muse et al. , 2007 ; Zeitlinger et al. , 2007 ) . 
These peaks have been attributed to promoter-proximal pausing based on several criteria ( Core and Lis , 2008 ; Muse et al. , 2007 ; Zeitlinger et al. , 2007 ) . 
In S. cerevisiae , promoter-proximal peaks occur only in stationary phase and by unknown mechanism ( Wade and Struhl , 2008 ) . 
All three types of mechanisms are well characterized in E. coli : promoter trapping ( Laishram and Gowrishan-kar , 2007 ; Rosenthal et al. , 2008 ) , promoter-proximal pausing ( Marr and Roberts , 2000 ; Hatoum and Roberts , 2008 ) , and attenuation ( Merino and Yanofsky , 2005 ) . 
Our findings establish that most promoter-proximal E. coli 
RNAP peaks correspond to ECs . 
First , the promoter-proximal RNAP peaks were offset in the direction of transcription by 150 bp ( Figure 4 ) . 
The transition from abortive to productive elongation , marked by release of s from promoter contacts 70 ( or from RNAP contacts ) , occurs within the first 20 nt of transcript elongation ( Chander et al. , 2007 ; Revyakin et al. , 2006 ) . 
Known cases of s - stimulated pausing in vivo occur no later than +25 70 ( Ring et al. , 1996 ) . 
Thus , the location of RNAP peaks at +150 is inconsistent with a block prior to promoter escape and EC formation . 
Second , NusA , which is thought to bind to ECs after release of s , and r , which requires > 50 nt of RNA to bind , 70 both appeared to be associated with RNAP in the promoterproximal peaks . 
Assuming that ChIP-chip captures a close-to-instantaneous snapshot of RNAP positions on DNA , we suggest that the promoter-proximal RNAP peaks reflect transcriptional attenuation caused by a mechanism other than r-dependent termination , rather than RNAP poised at promoters ( Wade and Struhl , 2008 ) . 
The position of these RNAP peaks is consistent with the typical position of transcription attenuators ( Merino and Yanofsky , 2005 ) and strongly resembles ChIP-chip profiles of RNAP on TUs known to be subject to transcriptional attenuation ( e.g. , trp and pyrBI ; Figure S7 ) . 
Promoter-proximal peaks in eukaryotes have been ascribed to paused ECs ( Core and Lis , 2008 ; Muse et al. , 2007 ; Zeitlinger et al. , 2007 ) . 
Although long elusive , transcription attenuation is now clearly shown to occur in eukaryotes ( Steinmetz et al. , 2006 ) . 
Conclusive evidence that promoterproximal halted RNAPs are actually paused rather than on a termination pathway exists only for a limited number of cases ( e.g. , Drosophila heat shock genes and bacteriophage l P 0 R ; Adelman et al. , 2005 ; Marr and Roberts , 2000 ) . 
The regulation of early elongation by attenuation may prove to be more 
For additional information, see the Supplemental Experimental Procedures.
Materials
E. coli K12 strains MG1655 and MG1655 HA3 : : nusG were used for all experiments . 
MG1655 HA3 : : nusG was constructed by gene replacement without selection to give a strain isogenic to MG1655 encoding three copies of the hemagglutinin ( HA ) epitope tag at the 50 end of nusG . 
Monoclonal antibodies against s ( 2G10 ) , RNAP ( anti-b 70 0 , NT73 or anti-b , NT63 ) , and NusA ( 1NA1 ) were purchased from Neoclone ( Madison , WI ) . 
The monoclonal 12CA5 antiHA antibody ( to target HA3 : : nusG ) was purchased from Roche . 
The polyclonal antibody against NusG was generated by Proteintech ( Chicago ) , and polyclonal antibody against r was a kind gift from Jeff Roberts ( Cornell University ) After labeling , ChIP samples were hybridized to a custom microarray from Nimblegen ( Madison , WI ) that contains two copies of 187,204 Tm-matched R45-mer oligonucleotides that tile the E. coli chromosome with an average of spacing of 24.5 bp . 
Cell Growth and ChIP-chip
Cells were grown in defined minimal medium ( with 0.2 % glucose ) with vigorous shaking at 37 C to mid-log ( light scattering at 600 nm equivalent to 0.4 OD ) . 
Formaldehyde was added to 1 % final , and shaking was continued for 5 min before quenching with glycine . 
Cells were harvested , washed with 
PBS , and stored at 80 C. Cells were sonicated and digested with micrococcal nuclease and RNase A before immunoprecipitation . 
The ChIP DNA sample was amplified by ligation-mediated PCR ( Lee et al. , 2006 ) to yield > 4 mg of DNA , pooled with two other independent samples , and sent to Nimblegen , where samples were labeled with Cy3 and Cy5 fluorescent dyes ( one for the ChIP sample and one for a control input sample ) and hybridized to a single microarray as a two-color experiment . 
ACCESSION NUMBERS
Raw microarray data have been deposited in GEO under the accession number GSE13938 . 
The Supplemental Data include Supplemental Experimental Procedures and seven figures and can be found with this article online at http://www.cell . 
com/molecular-cell/supplemental / S1097-2765 ( 08 ) 00891-5 . 
ACKNOWLEDGMENTS
We thank C. Herring for construction of the HA3 : : nusG allele , K. Struhl for helpful discussions and sharing results prior to publication , and J. Grass for assistance with a control experiment . 
This work was supported by grants to A.Z.A. ( USDA Hatch ) and R.L. ( NIH GM38660 ) . 
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