Initial transcribed region sequences influence the composition and functional properties of the bacterial elongation complex

Abstract

The bacterial RNA polymerase (RNAP) holoenzyme consists of a catalytic core enzyme (a 2 bb'v) in complex with a s factor that is essential for promoter recognition and transcription initiation. During early elongation, the stability of interactions between s and the remainder of the transcription complex decreases. Nevertheless, there is no mechanistic requirement for release of s upon the transition to elongation. Furthermore, s can remain associated with RNAP during transcription elongation and influence regulatory events that occur during transcription elongation. Here we demonstrate that promoter-like DNA sequence elements within the initial transcribed region that are known to induce early elongation pausing through sequence-specific interactions with s also function to increase the s content of downstream elongation complexes. Our findings establish s-dependent pausing as a mechanism by which initial transcribed region sequences can influence the composition and functional properties of the transcription elongation complex over distances of at least 700 base pairs.[Keywords: RNA polymerase; s factor; transcription initiation; promoter-proximal pausing; transcription elongation complex] Supplemental material is available for this article. The s subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation (Gross et al. 1998). When complexed with the RNAP core enzyme (subunit structure a 2 bb9v), different s factors specify the recognition of different classes of promoters (Gruber and Gross 2003). The primary s factor in Escherichia coli, s 70 , typically directs transcription initiation from promoters defined by two conserved hexameric DNA sequence elements, termed the À10 and À35 elements for their relationship to the transcription start site (position +1). During the transition from transcription initiation to transcription elongation, the growth of the nascent RNA destabilizes the interaction between s 70 and the RNAP core enzyme (Mekler et al. 2002;Murakami et al. 2002;Vassylyev et al. 2002;Nickels et al. 2005), but the complete release of s 70 is not required for entry into the elongation phase of transcription (Ring et al. 1996;Bar-Nahum and Nudler 2001;Mukhopadhyay et al. 2001;Mooney et al. 2005). Thus, although historically defined as an initiation factor, s 70 can also remain associated with the transcription elongation complex and influence the transcription process during elongation. A functional role for s 70 during elongation was first established in the context of bacteriophage l late gene transcription (for review, see Roberts et al. 1998). Specifically, the expression of the phage late genes under the control of the lQ anti-terminator protein depends on a s 70 -dependent pause that occurs during early elongation, shortly after the s 70 -containing RNAP holoenzyme has escaped the late promoter P R9 . Formation of this paused early elongation complex, which contains a stably bound nascent RNA that is 16 or 17 nucleotides (nt) in length, depends on an int...

Transcription Factor GreA Contributes to Resolving Promoter-Proximal Pausing of RNA Polymerase in Bacillus subtilis Cells

“…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.…”

“…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 Ϫ10-like sequences located downstream of the core sequence after promoter escape (6,11,21). The data obtained from in vivo KMnO 4 mapping suggest that E. coli RNAP stalls at the promoter-proximal regions in 10 to 20% of promoters, and GreA reduces the duration time of stalling at these regions for several genes (11).…”

Bacterial RNA polymerase can retain σ 70 throughout transcription

“…Some σ subunits release from core RNAP immediately upon the initiation of RNA synthesis (3). In contrast, the primary σ factor in Escherichia coli, σ 70 , may be associated in vivo with a fraction of transcription elongation complexes (TECs) even far downstream of the promoter (4)(5)(6)(7)(8). It is unclear whether this downstream association in vivo reflects retention of the initiating σ 70 subunit or binding of σ 70 after TEC formation and whether the σ 70 -TEC association is kinetically stable during transcript elongation (9).…”

Investigating transcription reinitiation through in vitro approaches

“…48 By showing that transcription-dependent loss of σ can be negatively modulated, previous studies have suggested the possibility that regulating the extent and time of σ release during elongation can influence not only elongation, but also RNA polymerase recycling and thus reinitiation. 49,50 Other in vitro studies have also shown that proteins as diverse as RapA, a bacterial homolog of eukaryotic SWI/ SNF proteins, and the ribosomal protein S1 can enhance transcription reinitiation, probably by counteracting inhibitory RNA polymerase-nucleic acid interactions, thus favoring polymerase recycling. 48,51 By contrast, transcription reinitiation could be thoroughly addressed in Archaea thanks to the availability of an in vitro system, reconstituted from purified Pyrococcus furiosus RNA polymerase and recombinant TBP and TFB, able to support multiple rounds of specifically initiated and terminated transcription.…”