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 ...

We have compared the activities of the RNA genomes of Pseudomonas aeruginosa phage PP7 and coliphages Q,B and f2 in a cell-free amino acid incorporating system derived from Escherichia coli. The rate of incorporation of