The transcription-repair coupling factor (TRCF, the product of the mfd gene) is a widely conserved bacterial protein that mediates transcription-coupled DNA repair. TRCF uses its ATP-dependent DNA translocase activity to remove transcription complexes stalled at sites of DNA damage, and stimulates repair by recruiting components of the nucleotide excision repair pathway to the site. A protein/protein interaction between TRCF and the β-subunit of RNA polymerase (RNAP) is essential for TRCF function. CarD (also called CdnL), an essential regulator of rRNA transcription in Mycobacterium tuberculosis, shares a homologous RNAP interacting domain with TRCF and also interacts with the RNAP β-subunit. We determined the 2.9-Å resolution X-ray crystal structure of the RNAP interacting domain of TRCF complexed with the RNAP-β1 domain, which harbors the TRCF interaction determinants. The structure reveals details of the TRCF/RNAP protein/protein interface, providing a basis for the design and interpretation of experiments probing TRCF, and by homology CarD, function and interactions with the RNAP.

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...

During transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded ‘transcription bubble,’ and selects a transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA ‘scrunching’ and ‘anti-scrunching,’ the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the transcription bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.

Pausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a "stepping" mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a "scrunching" mechanism, has not. We report a method that directly defines RNAP-active-center position relative to DNA in vivo with single-nucleotide resolution (XACT-seq; crosslink-between-active-center-and-template sequencing). We apply this method to detect and quantify pausing in initial transcription at 4 11 (~4,000,000) promoter sequences in vivo, in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4-5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard-coded by sequence elements similar to those for transcription-elongation pausing..

In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific protein-DNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway "backtracked" states that are substrates for the transcript cleavage factors of the Gre family, and on-pathway "scrunched" states that mediate pause escape. Here, using site-specific protein-DNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR' promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 2-4 bp, and that σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 2-3 bp. Analogous experiments with a library of 47 (~16,000) transcribed-region sequences show that the state scrunched by 2-3 bp--and only that state--is associated with the consensus sequence, T-3N-2Y-1G+1, (where -1 corresponds to the position of the RNA 3' end), which is identical to the consensus for pausing in initial transcription, and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T-3 resides in the DNA nontemplate strand. A cryo-EM structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T-3 of the consensus sequence exerts its effects by facilitating scrunching.