Probing the structure of Nun transcription arrest factor bound to RNA polymerase

Abstract

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5′ and the 3′ ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3′ end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5′ end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.Nun protein | Gre factors | transcription arrest | phage HK022 | ternary elongation complex T ranscription elongation by RNA polymerase (RNAP) is regulated by pausing and termination signals encoded in the transcribed DNA sequence, as well as by numerous protein factors and free RNA molecules. In transcribing ternary elongation complexes (TEC) (Fig. 1A), the RNA product remains transiently bound to a template DNA strand, forming a RNA:DNA hybrid 9-to 10-bp long (1). Transcription, although processive, is frequently interrupted by RNAP pausing. Mechanisms of such pausing include intrinsic pausing (2, 3) and backtracking (1, 4, 5), the process by which RNAP reverse-translocates. During backtracking, the RNA 3′ terminus moves from the catalytic center into the secondary channel, thus rendering TEC catalytically inactive. Bacterial transcription factors (TFs) GreA and GreB (6-10), as well as the analogous eukaryotic TFIIS factor (11, 12), rescue backtracked TEC by promoting degradation of this disengaged RNA, thus restoring the 3′ end of the transcript within the RNAP active center and allowing transcription to proceed.The 12-kDa Nun protein of coliphage HK022 excludes phage λ superinfection by specifically blocking TEC translocation on λ DNA (13,14). Nun competes with the λ N protein, which, conversely, enhances elongation and suppresses transcription termination. The amino-terminal RNA-binding domain (arginine-rich motif, or ARM) (Fig. 1B) is similar and plays the same role for both proteins: that of binding to the λ nascent transcript NUT sequence. The carboxylterminal domain (CTD) of Nun or λ N interacts with RNAP to block or stimulate transcription elongation, respectively (13). At high Nun concentrations, however, transcription arrest occurs in vitro or in vivo on templates lacking the λ NUT sequence (14, 15). Interaction between the Nun CTD and RNAP was probed using several different approaches. The CTD conjugated to a photoreactive group cross-links with a DNA template ...

Mfd Dynamically Regulates Transcription via a Release and Catch-Up Mechanism

“…However, there is growing evidence that Mfd has broader roles in resolving roadblocks encountered by RNAP well beyond TCR (Deaconescu et al, 2012; Deaconescu and Suhanovsky, 2017; Selby, 2017). Many different types of roadblocks can stall RNAP, including DNA lesions that are not targets of TCR (Smith and Savery, 2008), protein or protein complexes bound in front of the RNAP (Belitsky and Sonenshein, 2011; Zalieckas et al, 1998), or factors that interact with the RNA transcript to arrest RNAP (Mustaev et al, 2016). Mfd helps to resolve these conflicts by facilitating transcription through these roadblocks or terminating transcription.…”

Structural basis of transcription arrest by coliphage HK022 Nun in an Escherichia coli RNA polymerase elongation complex

“…Prior crosslinking analyses left open the possibility of a Nun:TEC stoichiometry of 1:1 or 2:1 ( Mustaev et al, 2016 ). To address this, we performed native mass spectrometry analysis of the TEC with and without addition of Nun.…”

“…A segment of Nun encompassing residues 44 to the C-terminus (residue 109) was found to crosslink to both the 5’- and 3’-ends of RNA transcripts ( Mustaev et al, 2016 ). Nun 44-109 crosslinks to the RNA 5’-end when the crosslinking moiety is placed at the -9 to -14 positions.…”

Transcription Elongation

“…Therefore, the role of GreB is thought to be to rescue arrested complexes. Mustaev et al 39 have proposed a model in which the bulky GreA Lys43 residue, which is stabilized by a salt-bridge with bE565, blocks RNA exit (Fig. 5).…”

“…79 A recent paper reveals additional important features of the Nun/TEC interaction. 39 Nun crosslinks RNA in an RNA:DNA hybrid within TEC. Both the 5 and the 3 0 ends of the RNA crosslink Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center.…”