Newly Discovered Antiterminator RNAs in Bacteriophage

Abstract

Antiterminator RNA directly modifies the transcription elongation complex so that it terminates less efficiently at intrinsic and factor-dependent terminators. These unusual RNAs were first discovered in bacteriophage HK022, where the nascent transcripts of the phage put sites promote full expression of phage genes during lytic infection. The activity of antiterminator RNA depends on specific structural elements that form as the transcript exits RNA polymerase. To further our understanding of the critical sequence features that permit RNA to serve as a transcriptional antiterminator, we have identified eight antiterminator RNA sequences in bacteriophages or prophages. There is strong sequence conservation among most of the put sequences, but sequence divergence is tolerated if critical structural elements are preserved. The most diverged antiterminator RNA is found in bacteriophage HK639. The HK639 putL transcript is an efficient antiterminator, and it has a novel structural feature that is critical for its activity. HK639 also displays a unique pattern of sensitivity to amino acid substitutions in the ␤ subunit zinc binding domain of RNA polymerase, adding to existing evidence that this domain interacts specifically with antiterminator RNA.Persistent transcription antitermination was first discovered in bacteriophage , and it is a common regulatory mechanism found in members of the phage family (reviewed in references 12, 23, and 35). In , full expression of most of the early genes requires the binding of an RNA binding protein encoded by the N gene to specific sites called nut, located in the early nascent transcripts. The N-nut complex modifies RNA polymerase so that pausing and termination at downstream sites is suppressed. Several host-encoded proteins stabilize the N-nut-RNA polymerase complex through protein-protein interactions (25). Bacteriophage HK022 is a member of the family of phages and also uses processive antitermination to transcribe most of its early genes (36). However, the modification of the elongating RNA polymerase is accomplished in a surprisingly different way. No protein, other than RNA polymerase, is required for antitermination (15,21). This factorindependent antitermination mechanism is mediated by the transcripts of two closely related cis-acting phage sites, putL and putR (polymerase utilization, left and right, respectively), that directly modify RNA polymerase so that it reads through downstream terminators with increased efficiency (15, 16). The put transcripts of HK022 are predicted to form similar structures, each consisting of two stem-loops separated by an unpaired base (2, 15). The structures differ from each other in the lengths of the stems and the location, composition, and size of various bulges and loops, but they have nearly identical antitermination activities. The proposed put RNA structure is based on computer modeling, genetic studies, and enzymatic structure-probing experiments on in vitro-synthesized put RNA. Nevertheless, our understanding of the functionally a...

Structural basis of transcriptional regulation by a nascent RNA element, HK022 putRNA

“…3d , Supplementary Fig. 10a ) 30 , 31 . Among the twenty-three mutations we generated, eleven mutants showed ≤ 20% anti-pausing activity (named ‘inactivating’ mutations) and three mutants showed ≥ 90% anti-pausing activity (named ‘inert’ mutations).…”