Transcription termination at intrinsic terminators: the role of the RNA hairpin.

Abstract

Intrinsic termination of transcription in Escherichia coli involves the formation of an RNA hairpin in the nascent RNA. This hairpin plays a central role in the release of the transcript and polymerase at intrinsic termination sites on the DNA template. We have created variants of the AtR2 terminator hairpin and examined the relationship between the structure and stability of this hairpin and the template positions and efficiencies of termination. The results were used to test the simple nucleic acid destabilization model of Yager and von Hippel and showed that this model must be modified to provide a distinct role for the rU-rich sequence in the nascent RNA, since a perfect palindromic sequence that is sufficiently long to form an RNA hairpin that could destabilize the entire putative 12-bp RNADNA hybrid does not trigger termination at the expected positions. Rather, our results show that both a stable terminator hairpin and the run of 6-8 rU residues that immediately follows are required for effective intrinsic termination and that termination occurs at specific and invariant template positions relative to these two components. Possible structural or kinetic modifications of the simple model are proposed in the light of these findings and of recent results implicating "inchworming" and possible conformational heterogeneity of transcription complexes in intrinsic termination. Thus, these findings argue that the structure and dimensions of the hairpin are important determinants of the termination-elongation decision and suggest that a complete mechanism is likely to involve specific interactions of the polymerase, the RNA terminator hairpin, and, perhaps, the dT-rich template sequence that codes for the run of rU residues at the 3' end of the nascent transcript.Transcription in Escherichia coli is terminated by two distinct mechanisms. One depends on rho, a hexameric protein that binds to the nascent RNA of the transcription complex and releases it at defined rho-dependent terminators along the template. The other, called intrinsic (or rho-independent) termination, also occurs at specific sites but depends only on signals encoded in the template. Intrinsic terminators all share a dyadic sequence that codes for a stable RNA hairpin followed by a string of uridine residues at the 3' end of the terminated RNA.Transcript elongation and transcript termination (followed by RNA release) represent alternative and kinetically competitive pathways for transcription at each position along the DNA template. This view of the elongation-termination decision has been formulated quantitatively in terms of competitive free energy of activation barriers (1) It has been shown for the AtR2 terminator (2) and confirmed for the AtR' terminator (Rees et al., unpublished data) that the termination pathway becomes accessible at intrinsic terminators because the transcription complex is massively destabilized in the vicinity of these sites. This provides a "zone of opportunity" for termination along the template, within which th...

Structural basis for RNA 3′-end recognition by Hfq

“…1A) is a small model sRNA that is induced in response to envelope stress and targets several outer membrane protein mRNAs, ultimately leading to their destabilization and degradation. As a typical sRNA, RybB is an independent, unprocessed bacterial transcription unit that consists of a 5′-terminal mRNA targeting region (23) followed by the RNA "body" that ends in a hairpin followed by a 3′-terminal oligo-U stretch as a consequence of intrinsic, Rho-independent transcription termination in bacteria (21).…”

“…This result helps to explain the selectivity of Hfq for many sRNAs, because U-rich RNA 3′ ends are common to sRNAs as a consequence of Rho-independent transcription termination (21). Furthermore, the sequestration of the RNA 3′ hydroxyl group within Hfq suggests a protection of the respective 3′ end from enzymatic modification with important implications for Hfq function.…”

“…From a physiological point-of-view, the high affinity of Hfq for U-rich RNA 3′ ends is intriguing because it represents a molecular feature shared by the large majority of sRNAs as a consequence of Rho-independent transcription termination (21). To determine whether the RNA 3′ end of our initial model sRNA, RybB, is indeed recognized by the proximal RNA-binding site of Hfq we set up a direct competition experiment.…”

Ribosomal protein S1 promotes transcriptional cycling

“…Inhibitory RNA polymerase-transcript interactions present a potential obstacle for continuous transcriptional cycling, with each individual cycle including transcript initiation, elongation, and termination. A number of studies have indicated that elongating E. coli RNA polymerase-transcript complexes are very stable but are destabilized at specific transcription terminators (Berlin and Yanofsky 1983;Wilson and von Hippel 1995;Mooney et al 1998;Gusarov and Nudler 1999;Yarnell and Roberts 1999). The hexameric ATP-dependent factor Rho has been shown to promote transcript release from the polymerase at certain terminators (Richardson and Conaway 1980;Shigesada and Wu 1980; for review, see Richardson 2002Richardson , 2003.…”

Modeling Classic Attenuation Regulation of Gene Expression in Bacteria

“…Let ω be a hairpin consisting of a handle without bulges and a negligibly small loop, with handle length h (the number of its constituent complementary base pairs). Four experimental points are known from [52]: 7; 0.11 , 8; 0.4 , 9; 0.54 , 12; 0.2 , where the first entry in the pairs is handle length h and the last one is premature termination probability within a U-run of length N . We will now derive an equation to describe premature termination probability in the form 1 ( ) P N − , where ( ) P N is probability of the polymerase reaching the end of N bases-long U-run without a slippage event.…”