Subcellular partitioning of transcription factors in Bacillus subtilis.
RNA polymerase (RNAP) requires the interaction of various transcription elongation factors to efficiently transcribe RNA. During transcription of rRNA operons, RNAP forms highly processive antitermination complexes by interacting with NusA, NusB, NusG, NusE, and possibly several unidentified factors to increase elongation rates to around twice those observed for mRNA. In previous work we used cytological assays with Bacillus subtilis to identify the major sites of rRNA synthesis within the cell, which are called transcription foci. Using this cytological assay, in conjunction with both quantitative native polyacrylamide gel electrophoresis and Western blotting, we investigated the total protein levels and the ratios of NusB and NusG to RNAP in both antitermination and mRNA transcription complexes. We determined that the ratio of RNAP to NusG was 1:1 in both antitermination and mRNA transcription complexes, suggesting that NusG plays important regulatory roles in both complexes. A ratio of NusB to RNAP of 1:1 was calculated for antitermination complexes with just a 0.3:1 ratio in mRNA complexes, suggesting that NusB is restricted to antitermination complexes. We also investigated the cellular abundance and subcellular localization of transcription restart factor GreA. We found no evidence which suggests that GreA is involved in antitermination complex formation and that it has a cellular abundance which is around twice that of RNAP. Surprisingly, we found that the vast majority of GreA is associated with RNAP, suggesting that there is more than one binding site for GreA on RNAP. These results indicate that transcription elongation complexes are highly dynamic and are differentially segregated within the nucleoid according to their functions.
Doherty
Geoff P
GP
School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
Meredith
Donna H
DH
Lewis
Peter J
PJ
D001412:Bacillus subtilis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002701:Chloramphenicol / Q000494:pharmacology; D003470:Culture Media; D017931:DNA Primers; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D013347:Subcellular Fractions / Q000648:ultrastructure; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification*; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; D003470:Culture Media; D017931:DNA Primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D002701:Chloramphenicol