Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase
Bacterial Gre factors associate with RNA polymerase ( RNAP ) and stimulate intrinsic cleavage of the nascent transcript at the active site of the enzyme ( 12 ) . 
In eukaryotic cells , the transcription factor , TFIIS , exerts similar activity ( 9 ) , indicating that Gre function is evolutionarily conserved in multisubunit RNAPs ( 9 ) . 
Gre factors consist of an N-termi-nal extended coiled-coil domain ( NTD ) and C-terminal globular domain ( CTD ) ( 19 , 32 ) . 
Escherichia coli possesses two highly homologous Gre factors : GreA and GreB . 
A structural study on the RNAP-GreB complex further revealed that CTD binds to the rim of the secondary channel of RNAP through which substrate nucleoside triphosphates for RNA synthesis enter the catalytic site ( 18 , 25 , 38 ) , while NTD extends into the secondary channel and the tip reaches the catalytic center ( 28 ) . 
Two acidic residues , D41 and E44 , located at the tip of NTD , are conserved in Gre factors , including those of Bacillus subtilis , and are proposed to assist RNAP function by coordinating the Mg2 ion and water molecule required for catalysis of RNA hydrolysis ( 20 , 28 , 31 ) . 
During the elongation process of transcription , roadblocks generated by DNA-binding proteins or specific DNA sequences induce RNAP to slide backward along the template ( backtrack ) , resulting in extrusion of the 3 terminus of nascent RNA through the RNAP secondary channel ( 9 ) . 
Several bio-in Bacillus subtilis Cells † Yoko Kusuya ,1 Ken Kurokawa ,2 Shu Ishikawa ,1 Naotake Ogasawara ,1 and Taku Oshima1 * Graduate School of Information Science , Nara Institute of Science and Technology , 8916-5 Takayama , Ikoma , Nara 630-0192 , Japan ,1 and Graduate School of Bioscience and Biotechnology , Tokyo Institute of Technology , 4259 Nagatsuta , Midori , Yokohama , Kanagawa 226-8501 , Japan2 
* Corresponding author . 
Mailing address : Graduate School of Information Science , Nara Institute of Science and Technology , 8916-5 , Takayama , Ikoma , Nara 630-0192 , Japan . 
Phone : 81-743-72-5430 . 
Fax : 81-743-72-5439 . 
E-mail : taku@bs.naist.jp . 
† Supplemental material for this article may be found at http://jb . 
asm.org / . 
Published ahead of print on 22 April 2011 . 
chemical and genetic studies have confirmed that the Gre factor facilitates endonucleolytic cleavage of extruded RNA to generate a new terminus that can be extended by RNAP , thus preventing transcription arrest during elongation and enhancing transcription fidelity ( 3 , 8 , 21 , 27 , 36 ) . 
Furthermore , the Gre factor participates in the stimulation of promoter escape and the suppression of promoter-proximal pausing during the beginning of RNA synthesis in E. coli ( 1 , 11 , 13 , 21 , 33 -- 35 ) . 
A fraction of RNAP is anchored to the promoter after initiation of RNA synthesis via persistent binding of 70 to the core promoter sequence in some E. coli promoters , and Gre factors upregulate transcription initiation from these promoters . 
In addition , E. coli RNAP often binds and stalls at 10-like sequences located downstream of the core sequence after promoter escape ( 6 , 11 , 21 ) . 
The data obtained from in vivo KMnO4 mapping suggest that E. coli RNAP stalls at the pro-moter-proximal regions in 10 to 20 % of promoters , and GreA reduces the duration time of stalling at these regions for several genes ( 11 ) . 
Consistent with these findings on Gre involvement in initiation of transcription , recent microarray analyses revealed that GreA activates transcriptional initiation of 19 genes under normal growth conditions and an even larger number of genes upon overexpression ( 34 ) . 
The intracellular level of GreA increases upon SigE overexpression in E. coli , indicating that GreA is important for transcriptional regulation under stress , rather than normal growth conditions ( 34 ) . 
Although Gre factors are universally conserved in bacteria , current knowledge of their functions in bacterial species other than E. coli is limited . 
The gre gene is essential in Mycoplasma pneumoniae ( 14 ) . 
Gre factors are important for osmotolerance in Rhizobium tropici and Sinorhizobium meliloti ( 26 , 39 ) . 
Bacillus subtilis possess one Gre factor , designated GreA . 
168 trpC2 Pasteur stock 168rpoCHis 168 rpoC : : pMUTinHis rpoC 15 168sigAHis 168 sigA : : pMUTinHis sigA 15 168nusAHis 168 nusA : : pMUTinHis nusA 15 YK02 168 greA : : pMUTinHis greA This study YK03 168 greA : : spec This study YK04 168 greA : : spec This study rpoC : : pMUTinHis rpoC YK05 168 greA ( D44A ) : : cat This study YK06 168 greA ( D44A ) : : cat This study rpoC : : pMUTinHis rpoC a spec , spectinomycin resistance gene ; cat , chloramphenicol resistance gene . 
The transcription elongation factors NusA , NusB , and NusG are concentrated in specific regions of the nucleoid termed transcription foci , which represent major sites of rRNA synthesis in B. subtilis cells . 
In contrast , B. subtilis GreA localizes uniformly throughout the nucleoid , suggesting its constant association with RNAP synthesizing mRNA ( 5 , 7 ) . 
Recent studies have explored the trafficking of core RNAP and the transcription factors , i.e. , the main sigma factor ( E. coli 70 A and B. subtilis ) and elongation factor NusA , on the chromosomes of E. coli and B. subtilis using ChIP-chip and ChAP-chip ( chromatin affinity precipitation coupled with DNA microarray ) methods ( 15 , 22 , 29a ) . 
The results suggest that the sigma factor in the initiation complex of RNAP is replaced with NusA upon transition to the elongation complex . 
Furthermore , our group demonstrated that in contrast to E. coli RNAP , which often accumulates at the promoter-proximal region , B. subtilis RNAP is evenly distributed from the promoter to coding sequences , indicating that RNAP B. subtilis recruited to the promoter promptly leaves the promoter-prox-imal region without trapping or pausing to form the elongation complex ( 15 ) . 
In the present study , we extended the ChAP-chip analysis to visualize the distribution of B. subtilis GreA on the chromosome and examined the effects of GreA inactivation on trafficking of core RNAP . 
Our data indicate that GreA is uniformly distributed throughout the transcribed region ( from promoters to coding regions ) in association with core RNAP , and its inactivation induces accumulation of RNAP at many promoter or promoter-proximal regions . 
Accordingly , we propose that GreA is constantly associated with core RNAP during transcriptional initiation and elongation and resolves its stalling at the promoter or promoterproximal regions , resulting in even distribution of the polymer-ase throughout the transcribed region in B. subtilis cells . 
MATERIALS AND METHODS
Bacterial strains and plasmids . 
The bacterial strains and primers used in the present study are listed in Table 1 and in Table S1 in the supplemental material , respectively . 
To create a B. subtilis strain expressing C-terminal 12 His-tagged GreA ( YK02 ) , a fragment encompassing the 3 region of the greA gene ( except the stop codon ) was amplified by PCR from B. subtilis 168 chromosomal DNA using the greA.f-greA . 
r primer set , and cloned between the HindIII and XhoI sites of pMUTinHis ( 17 ) . 
The resultant plasmid was integrated into the B. subtilis chromosome via single crossover to generate the YK02 strain . 
Western blot analysis confirmed a similar level of expression as that of wild-type GreA ( see Fig . 
S1 in the supplemental material ) . 
It was difficult to determine the functionality of GreA-His , since the greA-deleted mutant showed no apparent phenotype . 
However , our results indicate that His-tagged GreA minimally retains binding ability to core RNAP ( see Fig. 4A ) . 
To generate a greA deletion mutant ( greA , YK03 ) , the spec resistance gene , including the promoter region , was amplified from the pJL62 plasmid ( 16 ) by using the specF and specR primers , and the 5 and 3 flanking regions of greA were amplified from B. subtilis chromosomal DNA by using the primer sets greAF1-greAR1 spec and greAF2 spec-greAR2 , respectively . 
The primers greAR1 spec and greAF2 spec contained a 20-bp sequence complementary to the specF and specR primer sequences , respectively , at the 5 end . 
The three resulting fragments were fused via PCR by using the greAF1-greAR2 primer set and integrated into the B. subtilis chromosome via homologous recombination through the 5 and 3 flanking regions . 
A strain expressing C-terminal histidine-tagged RpoC ( 168rpoCHis ) was transformed with chromosomal DNA of YK03 to obtain YK04 ( rpoC-his greA ) . 
The GreA-D44A strain [ greA ( D44A ) ] harboring a point mutation altering Asp44 of GreA to Ala ( YK05 ) was constructed by using PCR ( shown schematically in Fig. 1 ) . 
The chloramphenicol resistance gene with the terminator region was obtained from the pDLT3 plasmid ( 23 ) by using the rPCR-CmF2 and rPCR-CmR2 primer sets . 
The greA gene and its downstream region were amplified from B. subtilis 168 chromosomal DNA as two fragments by using the primer sets D44AgreA1F-D44greA1R and D44greA2F Cm-D44AgreA2R , respectively . 
Primers D44AgreA1F and D44AgreA2R introduced substitutions of several bases to give one amino acid change ( Asp44 to Ala ) of GreA and the recognition site of the restriction enzyme , ApaLI , used for the confirmation of the substitution in greA gene . 
The D44greA2F Cm primer contained a 22-bp sequence complementary to the rPCR-CmF2 primer at the 5 end . 
The region upstream of greA was amplified from B. subtilis 168 chromosomal DNA by using the primers D44greA3F and D44greA3R Cm . 
The D44greA3R Cm primer contained a 22-bp sequence complementary to the rPCR-CmR2 primer at the 5 end . 
The resulting four fragments were fused by PCR using the D44greA3F-D44greA1R primer set and integrated into the B. subtilis chromosome via homologous recombination with selection for chloramphenicol resistance . 
A strain expressing C-terminal histidine-tagged RpoC ( 168rpoCHis ) was transformed with chromosomal DNA of YK05 to generate the YK06 strain [ rpoC-his greA ( D44A ) ] . 
Pulldown purification of RNAP complexes . 
B. subtilis strains expressing histi-dine-tagged protein -- 168rpoCHis , 168sigAHis , 168nusAHis , and YK02 ( expressing His-tagged GreA ) -- were grown in 400 ml of Luria-Bertani ( LB ) me-dium containing erythromycin ( 0.5 g/ml ) under aerobic conditions at 37 °C until cultures reached an optical density at 600 nm ( OD600 ) of 0.4 . 
Each culture was treated with formaldehyde ( 1 % final concentration ) for 30 min at 37 °C . 
Cells were washed with Tris-buffered saline buffer ( pH 7.5 ) and stored at 80 °C . 
Affinity purification of RNAP complexes was performed according to a previously described procedure for ChAP-chip experiments ( 17 ) with the following modifications . 
Dithiothreitol was removed from the UT buffer , Dynabead Talon ( 50 l ; Invitrogen ) used instead of MagneHis beads , and elution of complexes from Dynabeads was performed twice with 400 l of elution buffer . 
Recovered RNAP complexes were heated at 95 °C for 30 min to remove cross-linking , and the appropriate amounts of proteins were separated by using a 5 to 20 % SDS-PAGE gradient gel , followed by transfer to polyvinylidene difluoride membrane ( GE Healthcare ) via electroblotting at 100 V for 1.5 h ( RpoC , SigA , and NusA ) or 4 h ( GreA ) . 
Western blotting was performed according to the instructions of the Amersham ECL Plus Western blotting detection system ( GE Healthcare ) using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG ( Bio-Rad ) . 
Mouse polyclonal anti-RpoB antibody was obtained from Neoclone , and rabbit polyclonal anti- A antibody was kindly provided by Fujio Kawamura ( 24 ) . 
Rabbit polyclonal anti-NusA and anti-GreA antibodies were prepared as described below . 
Preparation of anti-GreA or NusA peptide antibody . 
Peptides corresponding to residues 21 to 37 ( EGKQKLEQELEYLKTVK ) , 40 to 55 ( EVVERIKIARS FGDLS ) , and 141 to 157 ( TVQTPGGEMLVKIVKIS ) of GreA and to residues 57 to 73 ( RVFARKDVVDEVYDQRL ) , 228 to 244 ( EAGDRSKISVRTDDP DV ) , and 355 to 371 ( EDDEPLFTEPETAESDE ) of NusA were synthesized , and mixtures of three peptides were used to raise antisera against GreA or NusA in rabbits ( Sigma Genosys , Japan ) . 
Anti-GreA and anti-NusA peptide antibodies were subsequently purified from antiserum by using peptide affinity column chromatography ( Sigma Genosys ) . 
ChAP-chip analysis . 
The strains used for ChAP-chip analysis were cultivated in 400 ml of LB medium containing the appropriate antibiotic ( s ) -- specifically , erythromycin ( 0.25 or 0.5 g/ml ) , spectinomycin ( 50 g/ml ) , and chloramphen-icol ( 2.5 g/ml ) -- under aerobic conditions at 37 °C until cultures reached a 
OD600 of 0.4 . 
The procedure for ChAP fraction preparation was similar to that for pulldown purification of the RNAP complex , and the final volume of the elution fraction was 40 l. Cross-linked whole-cell extract fractions before pu-rification of RNAP in each experiment were used to prepare control DNA for ChAP-chip analysis . 
Protein-DNA cross-links were dissociated by heating over-night at 65 °C . 
DNA was subsequently purified by using QiaQuick ( Qiagen ) and eluted with 50 l of nuclease-free water ( Ambion ) . 
Random amplification and terminal labeling of DNA in whole-cell extracts or affinity-purified fractions and hybridization to the custom Affymetrix tiling chip were performed as described previously ( 17 ) . 
The signal intensities of DNA isolated from the affinity purification and whole-cell extract fractions before purification ( control DNA ) were adjusted to confer a signal average of 500 . 
The signal intensities of DNA in the affinity-purified fraction were divided by those of control DNA for quantitative estimation of the enrichment of DNA fragments by affinity purification ( 37 ) . 
The binding signals represented by the enrichment values were visualized along the genome coordinate by using the In Silico Mo-lecular Cloning Program , Array Edition ( In Silico Biology , Japan ) . 
All experiments were performed in duplicate . 
A Analysis of the TR of RNAP . 
The binding peaks were automatically detected ( 15 ) , with the threshold value set as 2.0 . 
We selected genes positioned immediately downstream of the A binding sites , removing those located divergently and sharing the same A binding sites . 
Consequently , we selected 268 genes with sufficient RNAP signal intensities ( 0.95 ) and lengths ( 150 bp ) for traveling ratio ( TR ) calculation ( 15 , 29a ) . 
Transcriptome analysis . 
Total RNA was purified from wild-type , YK03 , and YK05 strains cultured in 200 ml of LB medium at 37 °C under aerobic conditions to an OD600 of 0.4 . 
Synthesis of cDNA , terminal labeling , and hybridization to the custom Affymetrix tiling chip were performed as described previously ( 4 ) . 
The signal intensities of perfectly matched probes ( only ) were used in this analysis and were adjusted to confer a signal average of 500 . 
Data visualization was performed by the In Silico Molecular Cloning Program . 
The average signal intensities of probes in each gene were calculated , and 2,824 genes with average signal intensities of 100 in wild-type , greA , and GreA-D44A cells were used to search for genes that were up - or downregulated upon inactivation of GreA . 
Comparison of transcriptome between the wild type and each greA mutant was performed by four different combinations using duplicate data for each strain . 
Array data . 
Raw data ( CEL format ) from ChAP-chip and transcriptome experiments have been deposited in ArrayExpress under accession numbers E-MEXP-3056 and E-MEXP-3055 , respectively . 
RESULTS
Distribution of GreA on the B. subtilis chromosome . 
To visualize the genome-wide association of GreA with RNAP , we created a strain expressing GreA tagged with 12 histidines at the C terminus under the control of the original promoter o the chromosome . 
GreA-His-expressing cells were cultivated in LB medium under aerobic conditions and harvested at an OD600 of 0.4 , followed by ChAP-chip analysis , as described earlier ( 15 ) . 
In parallel , we performed ChAP-chip analysis of the core RNAP ( subunit ) , A , and NusA , as well as transcriptome analysis using cells cultured under similar conditions . 
Typical distributions of protein-binding and transcription signals are shown in Fig. 2 , and the complete data set is presented in Fig . 
S2 in the supplemental material . 
The core RNAP binding signals started from the transcription start site ( 5 edge of contiguous transcription signals ; gray line ) and were evenly distributed along the transcribed region ( Fig. 2A and B ) . 
The A signals were observed symmetrically at the transcription start site ( Fig. 2C ) , while the NusA signals started slightly downstream of the transcription start site and were distributed throughout the transcribed regions ( Fig. 2D ) . 
These features are consistent with our previous findings ( 15 ) . 
We observed that GreA signals were distributed along the transcribed regions ( Fig. 2E ) , a finding similar to those for core RNAP and NusA . 
However , absolute signal intensities were lower and background signals were higher than binding signals of other proteins , probably because of indirect interaction of GreA with DNA and/or lower accessibility of His tag in the GreA-RNAP complex . 
In addition , we found no regions where GreA signals are observed without RNAP signals . 
Furthermore , we detected genome-wide positive correlation between the RNAP and GreA binding signals ( r 0.86 , Fig. 3A ) in the coding regions , similar to NusA binding signals ( r 0.94 , Fig. 3B ) . 
These results suggest that GreA is constantly associated with the majority of core RNAP during transcription elongation in B. subtilis cells , which is consistent with the overlapping localization of RNAP-green fluorescent protein ( GFP ) and GreA-GFP fluorescence ( 7 ) . 
The reduced correlation of signal intensities between GreA and RNAP in the low-signal-intensity region in Fig. 3A would be caused by the higher background signals of GreA . 
GreA is involved in the initiation and elongation of RNAP complexes . 
Several biochemical and structural studies have established that sigma factor and NusA compete for the same binding surface of core RNAP ( 10 , 41 ) , while GreA associates with core RNAP at a different site ( secondary channel ) . 
This finding suggests that , in addition to association with the elongation complex of RNAP , Gre factor also interacts with the initiation complex of RNAP . 
In support of this hypothesis , start sites of the GreA binding signals appeared to shift to transcription start sites , compared to those of NusA signals ( Fig. 2 and see Fig . 
S2 in the supplemental material ) . 
To confirm GreA association with the RNAP initiation complex , we analyzed the composition of RNAP complexes with the pulldown assay using His-tagged RpoC , GreA , NusA , or A as bait . 
Strains expressing 12 His-tagged GreA , NusA , A , and RpoC were cultivated to an OD600 of 0.4 , and cellular proteins were cross-linked with formaldehyde . 
Subsequently , cross-linked protein complexes were purified with nickel magnetic beads , and proteins included within the purified complexes were fractionated by SDS-PAGE after the removal of cross-linking by heat treatment . 
For precise comparison of the amounts of components within each complex , protein mixtures containing similar concentrations of RpoB ( representing the amount of core RNAP ) were subjected to SDS-PAGE ( see greA ( Fig. 5E ) and GreA-D44A strains ( Fig. 5F ) disclose decreased TR values in the majority of genes examined and not specific genes . 
Our findings suggest that GreA inactivation results in the stalling of RNAP at the promoter or promoterproximal region , supporting its general involvement in stimulation of promoter escape or suppression of promoter-proxi-mal pausing in B. subtilis cells . 
Notably , the GreA-D44A mutation exerted a more significant effect on RNAP trafficking than GreA deletion . 
Thus , it appears that GreA activity in assisting nucleolytic cleavage activity of RNAP is essential to resolve stalling at the promoter or promoter-proximal regions . 
Furthermore , it is possible that the GreA-D44A protein retains the ability to bind RNAP , similar to the E. coli mutant protein , which interferes with the intrinsic nucleolytic activity of active RNAP ( 20 ) , although no direct evidence to support this theory has been obtained . 
GreA contributes to resolving the stall of A-RNAP . 
Next , we attempted to characterize the stalled RNAP complexes in GreA-inactivated cells , focusing on genes for which core RNAP peaks appeared clearly at the promoter or promoterproximal regions in greA and GreA-D44A cells . 
We selected genes whose TR values were reduced by more than 0.20 , followed by visual inspection to strictly define core RNAP accumulation in greA mutants . 
As a result , 13 genes were identified in greA cells ( aee Fig . 
S6 in the supplemental material , genes 1 to 13 ) and 34 genes in GreA-D44A cells ( see Fig . 
S6 in the supplemental material , genes 2 to 35 ) . 
Among these , 1 and 22 genes were reproducibly detected only in greA and GreA-D44A cells , respectively , and 12 genes reproducibly detected in both strains . 
We further examined whether these stalled complexes were A-RNAP or NusA-RNAP by searching for genes in which A and NusA peaks could be discriminated . 
We identified 10 genes ( marked by asterisks in Fig . 
S6 in the supplemental material ) in which A and NusA peaks overlapped to a lesser extent . 
In most of these genes , accumulated RNAP peaks overlapped with A peaks ( Fig. 6 ) , suggesting that the majority of RNAP peaks induced by GreA inactivation constitute the A-RNAP complex . 
The greA deletion and D44A substitution have little impact on the transcriptome . 
Finally , we investigated the impact of GreA inactivation on genome-wide transcriptional regulation in B. subtilis cells . 
Total RNA was prepared from wild-type greA , and GreA-D44A cells , cultivated in LB medium under aerobic conditions , and harvested at an OD600 of 0.4 , and transcriptome profiles were obtained by using the tiling chip used for the ChAP-chip experiments , as described earlier ( 4 ) . 
We selected 2,824 genes with average signal intensities of 100 in the coding regions in all three strains and generated scatter plots of their transcription signal intensities , as shown in Fig. 7 . 
Next , we searched for genes that are up - or downregulated by 2.8-fold ( i.e. , log2 1.5 ) in greA and GreA-D44A cells , compared to wild-type cells , with P values ( Student t test ) lower than 0.05 . 
As a result , 28 upregulated and 35 downregulated genes were identified ( see Table S3 in the supplemental material ) . 
Among the 28 upregulated genes , 17 genes were upregulated in both mutant strains , and 24 and 21 genes were upregulated in greA and GreA-D44A cells , respectively . 
Similarly , 15 genes were downregulated in both mutant strains , and 24 and 26 genes were downregulated in greA and GreA-D44A , respectively . 
Furthermore , we observed no correlation between changes in the transcription level and RNAP accumulation ( Fig. 7 ) . 
These results indicate that inactivation of GreA has a limited impact on the transcriptome , and these effects are not directly related to RNAP accumulation in the promoter or promoter-proximal regions . 
DISCUSSION
To our knowledge , this is the first report on genome-wide distribution analysis of the bacterial elongation factor , Gre . 
The cellular level of B. subtilis GreA is twice that of RNAP , and the majority of GreA associates with RNAP ( 7 ) . 
We have shown here that GreA is evenly distributed from the promoter to coding regions and overlaps with RNAP engaged in transcription in B. subtilis ( Fig. 2 and 3 and see Fig . 
S2 in the supplemental material ) . 
Gre factors were previously proposed to transiently associate with stalled RNAP ( 9 ) . 
However , our data strongly suggest that GreA is not specifically recruited to stalled RNAP . 
In addition , pulldown assays of the components of RNAP complexes demonstrated that GreA associates with not only with the elongation complex of RNAP ( NusA-RNAP ) but also the initiation complex ( A-RNAP ) ( Fig. 4 ) . 
However , although the copurification analysis suggests that His-tagged GreA minimally retains binding ability to core RNAP ( Fig. 4A ) , it is possible that the His tag addition affects some GreA function and/or its binding affinity to RNAP , and this requires further investigation . 
GreA inactivation had no clear effects on the distribution of elongating RNAPs but induced a genome-wide shift in TR values , a finding indicative of RNAP pausing at promoter or promoter-proximal regions ( Fig. 5 ) . 
Clear RNAP peaks were detected at the promoter or promoter-proximal regions of 35 genes in GreA-inactivated cells ( Fig. 6 and see Fig . 
S6 in the supplemental material ) . 
Furthermore , the majority of the induced RNAP peaks colocalized with A peaks , suggesting the accumulation of A-RNAP . 
In E. coli cells , Gre factors enhance promoter escape and suppress promoter-proximal pausing of A-RNAP ( 11 , 13 , 21 , 34 , 35 ) . 
Based on these findings , we propose that B. subtilis Gre factor plays a similar role during the initiation of RNA synthesis in many promoters or promoter proximal regions . 
Although the resolution of our ChAP-chip analysis did not permit discrimination of RNAP accumulation at promoters or promoter-proximal regions , we favor the possibility of accumulation at promoter-proximal pausing , since B. subtilis RNAP is known to form unstable open complexes and synthesize smaller amounts of abortive transcripts than E. coli RNAP ( 2 , 40 ) . 
Recently , it was reported that the pausing of RNAP in E. coli is induced by direct and sequence specific interactions of RNAP with promoter-like sequences ( 6 , 29 ) . 
However , we have not yet found any correlation between RNAP stalling and promoter-like sequences at the promoter-proximal regions in B. subtilis . 
Further in vitro analysis of the effects of GreA on transcription initiation by B. subtilis RNAP and bioinformatics studies on the signals inducing RNAP pausing at promoter-proximal sites are required to elucidate the molecular mechanism of RNAP accumulation in greA mutants . 
The RNAP accumulation observed in greA cells was also detected in GreA-D44A cells , supporting the hypothesis that Asp44 of the B. subtilis GreA is essential to resolve the pausing of RNAP through stimulation of nucleolytic cleavage activity of RNAP . 
Interestingly , the effects of the GreA-D44A mutation on RNAP pausing were more extensive than those of the greA mutation . 
The E. coli Gre protein mutated at D41 ( corresponding to D44 in B. subtilis GreA ) inhibits elongation of transcription in vitro ( 20 ) . 
Recently , overexpression of a yeast TFIIS mutant harboring substitutions of two amino acids that stimulate intrinsic nucleolytic activity of RNAP was found to be lethal in yeast cells ( 30 ) . 
Based on the collective results , we propose that the B. subtilis GreA-D44A protein retains the ability to bind RNAP , and this binding interferes with the intrinsic nucleolytic activity of RNAP , which remains active , even without stimulation by Gre . 
However , in contrast to the data obtained with yeast , growth defects were not observed upon expression of the GreA-D44A protein , suggesting that the pausing of RNAP during the initiation and elongation of transcription does not occur frequently in B. subtilis cells , and the problem of stalling may be resolved by ways other than cleavage of the extruded 3 terminus of nascent RNA such as , for example , collapse of the association of RNAP with the DNA template . 
In E. coli cells , GreA inactivation has direct and negative effects on the transcription initiation frequencies of a number of genes ( 34 ) . 
However , we could not establish a direct impact of GreA inactivation on the transcriptome in B. subtilis cells under normal growth ( LB medium and aerobic ) conditions , even though RNAP trapping or pausing at promoters or pro-moter-proximal regions was induced in many genes in mutant strains . 
These observations suggest that the trapping or pausing frequency is lower in B. subtilis cells , compared to that in E. coli , probably due to differences in the biochemical properties of the two RNAP types . 
Initiation complexes of B. subtilis RNAP are efficiently converted to elongation complexes in vitro and in vivo , compared to E. coli RNAP ( 2 , 15 , 29a , 40 ) . 
As in several other bacteria , GreA function may be essential for B. subtilis growth under stress conditions that induce frequent pausing of RNAP . 
However , the phenotypes of greA mutants under various conditions are yet to be established , and further studies are required to understand the biological importance of GreA in B. subtilis cells . 
ACKNOWLEDGMENTS
We are grateful to Hiroki Takahashi for the suggestion of the statistical analysis . 
This study was supported by a KAKENHI grant-in-aid for scientific research in the Priority Area `` Systems Genomics '' from the Ministry of Education , Culture , Sports , Science , and Technology of Japan .