Structural and mechanistic basis of σ-dependent transcriptional 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

Structural basis for the bacterial transcription-repair coupling factor/RNA polymerase interaction

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

Structural and mechanistic basis of σ-dependent transcriptional pausing

SignificanceThe paradigmatic example of factor-dependent pausing in transcription elongation is σ-dependent pausing, in which sequence-specific σ-DNA interaction with a - 10 element-like sequence in a transcribed region results in pausing of a σ-containing transcription elongation complex. It has been proposed that σ-dependent pausing involves DNA scrunching, and that sequences downstream of the -10 element-like sequence modulate DNA scrunching. Here, using site-specific protein-DNA photocrosslinking, high-throughput sequencing, and cryoelectron microscopy structure determination, we show directly that σ-dependent pausing involves DNA scrunching, we define a consensus sequence for formation of a stable scrunched paused complex that is identical to the consensus sequence for pausing in initial transcription, and we identify positions of DNA scrunching on DNA nontemplate and template strands. Our results illuminate the structural and mechanistic basis of σ-dependent transcriptional pausing