N-Independent leftward transcription in coliphage lambda: Deletions, insertions and new promoters bypassing termination functions

Abstract

Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.

RNA sequence and secondary structure requiresments for rho-dependent transcription termination

“…The more proximal site is longer (124 nucleotides); however the other site (74 nucleotides) might also be sufficient. Genetic evidence suggests that the 393 base pair ninL99 deletion removes a site required for rho action at downstream termination sites (38). This deletion removes all of the proximal single-stranded region, and a portion of the distal one; thus the genetic evidence appears to be in good agreement with the analysis of potential rho binding sites contained in this paper.…”

Natural RNA Polymerase Aptamers Regulate Transcription in E. coli

“…Rho is a ring-shaped hexameric RNA helicase that travels with elongating RNAP (Epshtein et al, 2010; Nudler and Gottesman, 2002; Skordalakes and Berger, 2003). To terminate transcription Rho binds to relatively unstructured nascent RNA ( rut sites), categorized as ≈ 80 nt long regions with high cytidine/low guanine content (Boudvillain et al, 2013; Ciampi, 2006; Epshtein et al, 2010; Hart and Roberts, 1991; Peters et al, 2011; Salstrom et al, 1979). For iRAPs the C-content median value is 32.8%, whereas G-content median value is 15.4% (Figure S2D and E), suggesting that they can in principle facilitate Rho loading.…”

Learning from the Leaders: Gene Regulation by the Transcription Termination Factor Rho

“…The distance between clefts necessitates a spacer of at least ~12–14 RNA residues between recognized YC dinucleotides [15, 16]. As a result of these requirements, targeted mRNAs tend to be rich in pyrimidines, especially cytidines, within a 60–90 nt window designated a R ho ut ilization ( rut ) site [17, 18]. It is generally assumed that rut sites need to be free of RNA secondary structures [19], although it is feasible that structured regions between sites of direct Rho-RNA binding could be “looped out” [20].…”