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 β' subunit zinc binding domain of RNA polymerase, adding to existing evidence that this domain interacts specifically with antiterminator RNA.

Fig. 1.

Maps of the HK75, HK022, φEt88, and HK639 immunity regions. The vertical arrows indicate the EcoRI sites used in the cloning of the HK75 and HK639 immunity regions. The HK75 putR sequence was not present in the immunity region clone but was subsequently identified from the genomic sequence and has been included here for clarity. The diagonal lines indicate the relative location of the put sequence in each phage. The region marked M3 is 89% identical to the M3 sequence of phage φ80 and is a homolog of the λ OOP RNA (6). The maps are not drawn to the same scale. Numbered genes indicate coding sequences with unknown functions. The size of the cloned fragments from HK75 and HK639 are 2,137 bp and 4,130 bp, respectively. The φEt88 and HK022 immunity region maps represent 2,288 bp and 3,797 bp, respectively. HK639 ORFs 32 and 34 have no significant similarities with the protein database. HK639 ORF 33 has some similarity to the XRE family of transcriptional regulators, and ORF 38 is similar to a hypothetical E. coli protein.

Fig. 2.

(A) DNA sequence alignment of the put sites from phages HK75, HK639, HK022, and φEt88 and prophage sequences from E. coli 055:H7 strain CB9615 and Enterobacter cloacae strains ATCC 13047 and NCTC 9394. The consensus sequence shown above the alignment represents the most common base at each position. Gaps were introduced to improve the alignment, and the numbering is for reference only. Bases that match the consensus are represented by dots. Base changes are highlighted according to the color-coding shown in the consensus. The arrows above the sequence indicate the approximate location of regions of dyad symmetry. The hyphenated arrow indicates the variability in the placement of an internal loop (see panel B). The alignment was generated using Geneious software. (B) Predicted secondary structures of selected put transcripts. The numbering indicates the distance relative to the predicted start site of transcription. Bases that differ from the DNA consensus (see panel A) are boxed. The arrows indicate the conservation of intrastrand pairing despite sequence divergence from the consensus. The predicted secondary structures of HK639 putL, E. cloacae put, and HK022 putR were chosen for secondary structure display because they show the greatest divergence from the HK022 putL sequence. All secondary structure predictions were generated using Mfold (39).