Single-stranded RNA Recognition by the Bacteriophage T4 Translational Repressor, RegA

Abstract

The T4 protein, RegA, is a translational repressor that blocks ribosome binding to multiple T4 messages by interacting with the mRNAs near their respective AUG start codons. Other than the AUG, there are no obvious similarities between the affected mRNAs. High affinity RNA ligands to RegA were isolated using SELEX (systematic evolution of ligands by exponential enrichment). The selected RNAs exhibited the consensus sequence 5-AAAAUUGUUAUGUAA-3. The AUG was invariant, suggesting that it is the primary effector of binding specificity. The UU immediately 5 to the AUG and the upstream poly(A) tract were highly conserved among the selected RNAs. Boundary and footprinting experiments are consistent with the consensus sequence defining the RegA-binding site. Interestingly, chemical modification and nuclease digestion data indicate that the RNA-binding site is single-stranded, as if RegA discriminates between targets based on their primary sequence, not their secondary structure. Minor variations from the consensus at positions other than the universally conserved AUG have little effect on RegA binding, but accumulation of mutations has a profound effect on the interaction. Comparison of the in vivo targets for RegA to the SELEX-generated consensus suggests a repression pattern whereby the translation of individual messages is sequentially halted until the least similarly affected message, the regA gene itself, is repressed.Translational regulation has been shown to be an important means for controlling gene expression in a variety of organisms, both prokaryotic (1, 2) and eukaryotic (3,4). One of the more interesting regulatory mechanisms involves the repression of translation caused by RNA-binding proteins interacting specifically with mRNAs. Many of these translational repressors function by directly competing for mRNA binding with ribosomes, thus decreasing the level of translational initiation (5). Among the well characterized repressors, the bacteriophage T4 translational repressor, RegA, is unusual in that it affects the translation of many independent messages.The expression of at least nine T4 genes is reduced in the latter stages of the phage life cycle by the autoregulated product of the regA gene (6). Transcription of these genes is not altered, and thus RegA-mediated repression occurs post-transcriptionally (7). Genetic analysis and in vitro footprinting indicate that RegA specifically interacts with several of the regulated mRNAs near their translational start sites (8 -10). The presence of RegA alters the binding of the 30 S subunit of Escherichia coli ribosomes to these mRNAs, thus preventing translational initiation (9). Taken together, these data are consistent with RegA altering gene expression in vivo by obstructing ribosome binding to specific mRNAs.Although RegA repression is specific, the mRNA sequences that are bound by the repressor display few similarities. A consensus for the region surrounding the translational start sites of the affected mRNAs is indistinguishable from one generated for all ...

Bacteriophage T4 Genome

“…The configuration suggests that repression is transmitted through translational coupling to gene 62 and regA itself. SELEX-isolated binding sites did not precisely match any specific T4 site, but the consensus sequence (5Ј-AAAAUUGUUAUGUAA-3Ј) resembles many of the RegA-sensitive RBS (113). Interestingly, RegA is conserved in all T-even-related phages examined, although it is nonessential under laboratory growth conditions.…”

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: Initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase

“…Specific RNA recognition by proteins underlies many biological processes that demand high-fidelity performance+ In the best-described examples, highly specific outcomes are the result of the recognition of RNA elements that comprise short sequence-specific features placed in a defined structural framework+ Thus, specific aminoacylation of transfer RNAs by the cognate aminoacyl-tRNA synthetase requires the accurate placement of a few identity nucleotides within the generic tRNA structure that is approximately 76 nt long (Pallanck et al+, 1995)+ Shorter elements (ca+ 20-30 nt long) recognized by combined structural and sequence information are the helix/loop combinations bound by the HIV Tat protein (Puglisi et al+, 1992), phage R17/MS2 coat protein (Valegård et al+, 1994), and U1A spliceosomal protein (Oubridge et al+, 1994)+ This is not the only way in which proteins recognize specific sites on RNA, however+ For instance, evidence exists that the phage T4 translational repressor, regA, recognizes a consensus sequence of some 12-15 linear nucleotides in unstructured RNA (Szewczak et al+, 1991;Brown et al+, 1997)+ Here, as a result of studying the replication of a positive strand RNA virus, we report a further strategy for specific RNA recognition that requires a combination of a very short specific-sequence and adjacent non-or low-specificity secondary structure+ Positive strand RNA viruses replicate via two sequential transcriptional steps that synthesize full-length minus and plus sense genomes; additionally, in some viruses, internal initiation on the minus strand (Miller et al+, 1985) results in the synthesis of subgenomic RNAs that are collinear with the 39-region of the genomic RNA and that serve as mRNAs expressing downstream cistrons (Buck, 1996)+ Specific initiation sites are used for each of these transcriptional events, and a breakdown of the fidelity of initiation site selection would lead to truncated genomes and viral proteins+ The cisrequired promoter elements controlling these specific strand initiations have been studied in a number of viruses by in vivo approaches using deleted genomes and by in vitro approaches using viral RNA-dependent RNA polymerase (RdRp) preparations (Buck, 1996)+ These studies have generally supported the view that accurate transcription is controlled by the detection of specific features of either sequence or a combination of sequence and structure (e+g+, Miller et al+, 1986;Dreher & Hall, 1988;Levis et al+, 1990;Cui & Porter, 1995;Song & Simon, 1995;Miranda et al+, 1997;Siegel et al+, 1997)+ Against this backdrop of apparently specific recognition of defined, discrete promoter elements, we have studied the features directing minus strand synthesis by the turnip yellow mosaic virus (TYMV) RdRp+ Minus strand synthesis initiates specifically opposite the penultimate resi...…”

In Vitro Genetic Analysis of the RNA Binding Site of Vigilin, a Multi-KH-Domain Protein

“…Although an early study (8) and several subsequent studies used DNA gel mobility shifts to separate free and protein-bound DNA, in vitro genetic selection with an RNA gel mobility shift has not been described. RNA binding sites have been identified for several proteins by using in vitro genetic analysis (11,12,15,31,39,56,62,63,66). Those studies used purified RNA binding proteins.…”