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.
Highlights
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]
Among the well characterized repressors, the bacteriophage T4 translational repressor, RegA, is unusual in that it affects the translation of many independent messages
The presence of RegA alters the binding of the 30 S subunit of Escherichia coli ribosomes to these mRNAs, preventing translational initiation [9]
Summary
Translational repression of several early T4 genes results from the binding of the regA gene product to specific mRNAs near their respective AUG start codons, preventing the initiation of translation [9]. A simple explanation for these results is that a single binding event occurs at lower RegA concentrations and that an additional protein binds to each ligand at higher concentrations yielding the second shift This is consistent with the sequence analysis above that suggests that two RegA-binding sites exist on each of these RNAs. The AUG of the putative second binding site was altered to UUU, and the 10 3Ј-most nucleotides were removed to test whether a single binding event could be observed. Between positions G44 and U59 in the full-length version of ligand A are completely protected from nuclease attack by both concentrations of repressor (Fig. 4A) This second RegA footprint covers the putative 3Ј-binding site of the RNA.
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