Jim Karam

Translational repression: Biological activity of plasmid-encoded bacteriophage T4 RegA protein

The RegA protein of bacteriophage T4 is a translational repressor that regulates expression of several phage early mRNAs. We have cloned wild-type and mutant alleles of the T4 regA gene under control of the heat-inducible, plasmid-borne leftward promoter (PL) of phage lambda. Expression of the cloned regA+ gene resulted in the synthesis of a protein that closely resembled phage-encoded RegA protein in biological properties. It repressed its own synthesis (autogenous translational control) as well as the synthesis of specific T4-encoded proteins that are known from other studies to be under RegA-mediated translational control. Cloned mutant alleles of regA exhibited derepressed synthesis of the mutant regA gene products and were ineffective in trans against RegA-sensitive mRNA targets. The effects of plasmid-encoded RegA proteins were also demonstrated in experiments using two compatible plasmids in uninfected Escherichia coli. The two-plasmid assays confirm the sensitivities of several cloned T4 genes to RegA-mediated translational repression and are well-suited for genetic analysis of RegA target sites. Repression specificity in this system was demonstrated by using wild-type and operator-constitutive translational initiation sites of T4 rIIB fused to lacZ. The results show that no additional T4 products are required for RegA-mediated translational repression. Additional evidence is provided for the proposal that uridine-rich mRNA sequences are preferred targets for the repressor. Surprisingly, plasmid-generated RegA protein represses the synthesis of some E. coli proteins and appears to enhance selectively the synthesis of others. The RegA protein may have multiple functions, and its binding sites are not restricted to phage mRNAs.

Genetic control of mRNA decay in T4 phage-infected Escherichia coli

Abstract Several T4 phage mutations were isolated that resulted in the hyperproduction of certain phage-induced proteins in infected Escherichia coli hosts. These mutations map in T4 gene reg A and affect a diffusable substance that plays a role in the control of structural and functional decay of a small number of mRNA species that are synthesized during early times after phage infection. The reg A gene product may either by a specific ribonuclease or may indirectly affect mRNA decay by influencing the selectivity of translation.

Expression of bacteriophage T4 genes 45, 44, and 62 I. Discoordinate synthesis of the T4 45- and 44-proteins

Abstract T4 genes 45, 44, and 62 code for proteins that play essential roles in phage DNA replication. These three genes map as a cluster on the T4 chromosome and are thought to be transcribed into one polycistronic mRNA. We reexamined the possibility that these three genes are cotranscribed and obtained results which suggested that the protein products of genes 44 and 62 are translated from one mRNA species, but that the protein products of genes 45 and 44 are translated from different mRNA species. Cotranscription of T4 genes 44 and 62 was implied by polar effects that two amber mutations in gene 44 exhibited on the synthesis of the protein product of gene 62. In contrast, an amber mutation in T4 gene 45 did not affect the synthesis of the protein products of genes 44 and 62, and the protein products of genes 45 and 44 were found to be disproportionately synthesized under more than one type of growth condition. In the accompanying paper, Bowles and Karam present genetic evidence for the existence of a regulatory site for gene 44 that does not service gene 45. This also suggests that genes 45 and 44 belong to different operons. The T4 gene 45 amber mutation that we used in our studies exhibited a cis-dominant effect on the biological activity (although not on the synthesis ) of a partially defective protein product of gene 44. The results suggest that the protein products of genes 45 and 44 are more likely to interact with each other in vivo when they are coded for by the same genome than when they are synthesized from genotypically different coinfecting genomes.

Expression of bacteriophage T4 genes 45, 44, and 62 II. A possible regulatory site between genes 45 and 44

Abstract A cis -acting T4 phage mutation was isolated that resulted in temperature-dependent hyperproduction of the protein products of phage genes 44 and 62. The mutation (named hp6 ), mapped between markers in T4 genes 45 and 44, did not affect T4 45-protein synthesis and may be an alteration of a regulatory region for T4 genes 44 and 62.