Suppression of Factor-Dependent TranscriptionTermination by AntiterminatorRNA

Abstract

Nascent transcripts of the phage HK022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. We show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial Rho protein and a second that depends on the HK022-encoded Nun protein. Suppression was efficient when the termination factors were present at physiological levels, but an increase in the intracellular concentration of Nun increased termination both in the presence and absence of put. put-mediated antitermination thus shows no apparent terminator specificity, suggesting that put inhibits a step that is common to termination at the different types of terminator.After initiating RNA synthesis, RNA polymerase (RNAP) continues to elongate the transcript until it reaches a termination site. At such sites, the enzyme has a high probability of dissociating from the transcript and the template (reviewed in references 26 and 32). Bacteria have two basic types of transcription termination signals, which differ in their requirements for halting elongation. Intrinsic terminators can stop transcription through the action of the nascent transcript. Formation of an RNA stem-loop immediately upstream of a U-rich stretch in nascent RNA disrupts RNA-DNA base pairs within the transcription elongation complex, and this destabilizes the complex (15,20,47). By contrast, factor-dependent terminators recruit a termination factor to the nascent transcript. Two termination factors have been well characterized: the bacterial Rho protein and the bacteriophage-encoded Nun protein. After binding to nascent transcripts, they both act on the nearby elongation complex. Rho has an ATP-driven RNA-DNA helicase activity, which is thought to destabilize the elongation complex (7,30). Nun is transferred from its RNA binding site to the elongation complex, where it is thought to anchor RNAP to the DNA template within a few hundred nucleotides downstream of the binding site (16,39,43). Dissociation of Nun-arrested polymerase from the template and the transcript has not been observed in vitro and appears to require an additional factor or factors. Recent evidence suggests that the Escherichia coli Mfd protein can stimulate the dissociation of Nun-arrested complexes (42).E. coli and its bacteriophages alter the efficiency of transcription termination in order to control the expression of genes located downstream of terminators (reviewed in reference 44). For example, the phage antitermination proteins N and Q modify RNAP so that it reads through intrinsic and rho-dependent terminators. Both N and Q recognize specific phage sequences (nut and qut, respectively) before they modify polymerase, and this limits antitermination to RNAP molecules that are transcribing phage DNA. Elongating RNAP that has been modified by interaction with either protein retains the modification as it translocates, as shown by its abilit...

Protection of antiterminator RNA by the transcript elongation complex

“…and T L4 (Limberger and Campbell, 1987), have been verified experimentally; the others have not been tested. Rho‐dependent terminators, which are not predicted by this program but which are also suppressed by put (King and Weisberg, 2003), could also be present.…”

Conservation and diversity in the immunity regions of wild phages with the immunity specificity of phage λ

“…In the IGR, patterns indicate the alleles found in λ, HK97, P22, or the hybrid site found in ST104. The IGR in HK022 is not functionally homologous to those in λ and related phages, as it contains an RNA sequence, put , which causes anti‐termination by a direct interaction with RNA polymerase (King and Weisberg, 2003). The location of regulatory sites in the λ sequence is shown (Friedman and Olson, 1983; Graham and Richardson, 1998; Richardson, 2002).…”

Transcription Termination and Antitermination of Bacterial CRISPR Arrays

“…Indeed, a targeted antitermination mechanism has been described for CRISPR array leader sequences in Pseudomonas aeruginosa (Lin et al, 2019). Alternatives to BoxA-mediated antitermination of CRISPR arrays could include antitermination by NusG homologues such as RfaH (Goodson et al, 2017; Goodson and Winkler, 2018; Kang et al, 2018), or inhibition of Rho activity by cis -acting RNA elements, similar to the Put element of phage HK022 (King and Weisberg, 2003). An alternative possibility is that CRISPR array processing by Cas6 may be so efficient in some bacterial species that the CRISPR array transcript is processed downstream of Rho, preventing Rho from catching RNAP on the RNA.…”