Com, the phage Mu mom translational activator, is a zinc-binding protein that binds specifically to its cognate mRNA

摘要

Bacteriophage Mu controls an unusual DNA-modification function encoded by the mom gene, which is located in an operon that consists of two overlapping genes. The corn gene, located proximal to the 5’ end of the common mRNA transcript, encodes a polypeptide of 62 amino acids that is required for translation of mom. Analysis of the derived amino acid sequence reveals that Com contains zinc-binding finger motifs, suggesting that Com may be a zinc-activated regulatory protein. Atomic absorption analysis showed that there is about one zinc bound per molecule of Com. We have subcloned the corn gene into an expression vector and thus have overproduced and purified the Com protein. By gel retardation analysis with various 32P-labeled RNAs (made by in vitro transcription with T7 RNA polymerase), we show that Com binds specifically to corn-morn mRNA. A single C + U substitution mutation, located 26 nucleotides upstream from the mom translation startcodon, abolishes Com binding. The nature of the Com target sequence was deduced from in vitro footprinting analyses. The results are consistent with the existence of a complex stem-loop structure within the overlap of the corn-morn open-reading-frames. Combinding to its target site results in the destabilization of a proposed translation-inhibitor stem-loop (TIS) to expose the Shine-Dalgarno sequence and mom translation initiation codon. This suggests that Com interaction with a specific site on its cognate mRNA alters the mRNA secondary structure to activate translation of mom.