Subcellular Partitioning of Transcription Factors inBacillus subtilis

Abstract

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.Unlike the situation in eukaryotes, a single RNA polymerase (RNAP) is responsible for carrying out transcription of all classes of genes in prokaryotes. As rRNA synthesis is the rate-determining step in ribosome assembly, the efficiency of this process determines the rate at which organisms can produce proteins and ultimately divide (23, 37). The rate of rRNA transcription has also been found to be around twice the rate of mRNA transcription (52), and this is due to the types of transcription elongation complexes (EC) formed during these two types of transcription. The vast majority of stable RNA (rRNA and tRNA) is produced by antitermination ECs that are resistant to transcription pause signals and ensure that full-length transcripts are efficiently produced (1, 11, 52), although transcription of tRNA genes that are located outside rRNA operons does not appear to depend on the same regulatory sequences that are responsible for the antitermination complex formation that occurs in rRNA operons (Doherty, unpublished data). mRNA is produced by ECs that tend to be highly susceptible to pausing, and this appears, among other things, to help ensure the close coupling of transcription and translation, transcription fidelity, to enhance responsiveness to transcription attenuators (5, ...

Transcription factor dynamics

“…This is due to the relatively low level of recruitment of RNAP to rRNA operons at low growth rates, when the demand for ribosomes is much less than at higher growth rates (Lewis et al, 2000). However, NusB is still required for antitermination complex formation at low growth rates, and so TF can still be observed under these conditions, making this a useful fusion for monitoring sites of rRNA synthesis at all growth rates (Doherty et al, 2006). Interestingly, we think the well-defined foci observed with the NusB-GFP fusion represent antitermination complexes loaded on individual rRNA operons or clusters.…”

“…The localization of these fusions is shown in Fig. 3, and all have been shown to be nucleoid associated (Davies et al, 2005;Doherty et al, 2006). However, they display vastly different patterns which reflect the biochemical roles they have been assigned.…”

“…3C), consistent with biochemical studies from E. coli and Mycobacterium tuberculosis suggesting that it is restricted to antitermination complex formation (Torres et al, 2004;Greive et al, 2005). Furthermore, a dual-labelled strain showed that these foci co-localize with RNAP (Doherty et al, 2006), and are rapidly reduced upon induction of the stringent response. One striking difference between NusB (and NusA to a lesser extent) and RNAP localization to TF is observed in slow-growing cells when NusB (and NusA) TF are still clearly visible, but they are almost totally absent with the RNAP-GFP fusion (Lewis et al, 2000;Davies et al, 2005;Doherty et al, 2006).…”

“…Furthermore, a dual-labelled strain showed that these foci co-localize with RNAP (Doherty et al, 2006), and are rapidly reduced upon induction of the stringent response. One striking difference between NusB (and NusA to a lesser extent) and RNAP localization to TF is observed in slow-growing cells when NusB (and NusA) TF are still clearly visible, but they are almost totally absent with the RNAP-GFP fusion (Lewis et al, 2000;Davies et al, 2005;Doherty et al, 2006). This is due to the relatively low level of recruitment of RNAP to rRNA operons at low growth rates, when the demand for ribosomes is much less than at higher growth rates (Lewis et al, 2000).…”

“…The transcription factors NusA, NusB, NusG and GreA are highly conserved throughout prokaryotes and are even present in the minimal genome of Mycoplasma genitalium. Using the same system as that used for tagging B. subtilis RNAP in vivo (Lewis & Marston, 1999;Lewis et al, 2000), functional GFP fusions were made to NusA, NusB, NusG and GreA and their subcellular localization patterns observed (Davies et al, 2005;Doherty et al, 2006). The localization of these fusions is shown in Fig.…”