RNA extension drives a stepwise displacement of an initiation-factor structural module in initial transcription

Abstract

All organisms--bacteria, archaea, and eukaryotes--have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription-initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription-initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate, and define details of, stepwise, RNA-extension-driven displacement of the "s finger" of the bacterial transcription initiation factor s. The structures reveal that--for both the primary s factor and extracytoplasmic (ECF) s factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA--the "s finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from ~5 nt to ~10 nt.

Anti-pausing activity of region 4 of the RNA polymerase σ subunit and its regulation by σ-remodeling factors

“…4D). Thus, the s3.2-finger should hinder the extension of RNAs longer than 5 nt and favor abortive initiation (Murakami et al, 2002) (Li et al, 2020). A model of the s3.2-finger deletion on the EcoRNAP structure ( Fig.…”

“…Indeed, the unstructured linker connecting domains s3 and s4 (formed by the s regions 3.2 and 4.1) is located in the RNA-exit channel and represents a barrier for growing RNA chains. This linker is displaced by RNA upon promoter escape (Li et al, 2020). A clash between the s3.2-finger and >4-nt RNA chains hinders RNA extension and may cause the formation of abortive RNAs, thus contributing to pausing during initial transcription (Murakami et al, 2002) (Basu et al, 2014) (Duchi et al, 2016) (Zhang et al, 2012)…”

The σ Subunit-Remodeling Factors: An Emerging Paradigms of Transcription Regulation

“…Domain σ4 interacts with the β-flap domain of core RNAP and harbors a helix-turn-helix DNA binding domain that recognizes the −35 motif. The σ3 and σ4 subunits are connected by a weakly structured linker (region 3.2) that fills the RNA exit channel and is ejected upon the initial RNA synthesis ( Zhang et al, 2012 ; Li et al, 2020 ). Most bacterial promoters recognized by group 1 and 2 σ subunits belong to the −10/−35 class and contain the −10 and also the −35 elements.…”

Kinetic-Mechanistic Evidence for WhichE. coliRNA Polymerase-λPROpen Promoter Complex Initiates and for Stepwise Disruption of Contacts in Bubble Collapse

“…Transcription initiation, including open complex formation and subsequent steps of NTP binding and synthesis of a RNA-DNA hybrid, is a prime target for regulation. An understanding of the kinetics and mechanism of transcription initiation (1-9) is needed to complement structural characterization of RNAP-promoter initiation complexes (10)(11)(12)(13), with applications to antibiotic development and synthetic promoter design (14). In the initial specific closed complex (RPC), the start site region of duplex promoter DNA is not contacted by RNAP (10,15).…”