Single-stranded RNA recognition by the bacteriophage T4 translational repressor, regA.
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.
Brown
D
D
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA.
Brown
J
J
Kang
C
C
Gold
L
L
Allen
P
P
D017122:Bacteriophage T4 / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D017931:DNA Primers; D007256:Information Systems; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; C066700:RegA protein, Enterobacteria phage T4; D014764:Viral Proteins; D012313:RNA