The bacteriophage T4 regA gene: primary sequence of a translational repressor (original) (raw)
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Bacteriophage T4 regA protein binds to mRNAs and prevents translation initiation
Proceedings of the National Academy of Sciences, 1987
The bacteriophage T4 regA protein is a translational repressor of a subset of phage mRNAs. We show here that purified regA protein binds specifically to target mRNAs near the initiating AUG and occludes binding of ribosomes. Translational repression by regA protein diminishes expression of many genes whose mRNA sequences around the initiating AUG codons are different. A comparison of nucleotide sequences from several regA-repressed mRNAs suggests that the initiating AUG is an important, but not sufficient, sequence for regA binding.
Crystal structure of the T4 regA translational regulator protein at 1.9 A resolution
Science, 1995
The translational regulator protein regA is encoded by the T4 bacteriophage and binds to a region of messenger RNA (mRNA) that includes the initiator codon. RegA is unusual in that it represses the translation of about 35 early T4 mRNAs but does not affect nearly 200 other mRNAs. The crystal structure of regA was determined at 1.9 A resolution; the protein was shown to have an a-helical core and two regions with antiparallel P sheets. One of these P sheets has four antiparallel strands and has some sequence homology to RNP-1 and RNP-2, which are believed to be RNA-binding motifs and are found in a number of known RNA-binding proteins. Structurally guided mutants may help to uncover the basis for this variety of RNA interaction.
Cloning, nucleotide sequence, and overexpression of the bacteriophage T4 regA gene
Proceedings of the National Academy of Sciences, 1985
The bacteriophage T4 regA gene codes for a regulatory protein that controls the expression of a number of T4 early genes, apparently at the level of translation. Restriction fragments containing the regA structural gene have been cloned into phage M13, and the nucleotide sequence has been determined. Translation of the DNA sequence predicted that regA protein contains 122 amino acids, with a Mr of 14,620. A DNA fragment carrying 85% of the coding sequence of regA has been cloned into the phage A leftward promoter PL expression vector pAS1, and a high level of truncated regA protein was produced by nalidixic acid induction. Protein chemical studies of the truncated regA protein gave results consistent with the nucleotide sequence of the regA gene. Subsequently, an intact regA gene was cloned into plasmid pAS1 and overexpressed. The regA protein produced in this way regulates the level of T4 45 and 44 proteins when their corresponding genes are carried on the same plasmid as the regA gene.
Analytical Biochemistry, 1999
To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein. In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His 6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole. The system was further modified to avoid cloning and the toxic effects of RegA on Escherichia coli by the polymerase chain reaction (PCR), producing linear templates with the configuration T7 promoter-TIR-regA-His 6 . A translation initiation region was used that conforms to consensus E. coli and eukaryotic initiation sites and eliminates the target for RegA autogenous repression. RegA69-His 6 synthesized in E. coli S30 or wheat germ extracts displayed RNAbinding properties similar to wild-type RB69 RegA. Specificity of RNA binding was demonstrated by in vitro repression of T4 gp44 and gp45 but not -lactamase, by differential binding to poly(U)-and poly(C)agarose, and by site-specific binding to a 23-base gene 44 target RNA but not to mutant 44 RNA. Therefore, addition of the His 6 tag to the C-terminus of RB69 RegA does not dramatically alter RNA binding, indicating that this region is not directly involved in site recognition. With access to several T4-like phage genomes and regA mutant sequences, in vitro synthesis of His-tagged proteins directly from linear PCR products provides a convenient and efficient system to study RegA and other interesting RNA-binding proteins.
DNA polymerase of bacteriophage T4 is an autogenous translational repressor
Proceedings of the National Academy of Sciences of the United States of America, 1988
In bacteriophage T4 the protein product of gene 43 (gp43) is a multifunctional DNA polymerase that is essential for replication of the phage genome. The protein harbors DNA-binding, deoxyribonucleotide-binding, DNA-synthesizing (polymerase) and 3'-exonucleolytic (editing) activities as well as a capacity to interact with several other T4-induced replication enzymes. In addition, the T4 gp43 is a repressor of its own synthesis in vivo. We show here that this protein is an autogenous repressor of translation, and we have localized its RNA-binding sequence (translational operator) to the translation initiation domain of gene 43 mRNA. This mechanism for regulation of T4 DNA polymerase expression underscores the ubiquity of translational repression in the control of T4 DNA replication. Many T4 DNA polymerase accessory proteins and nucleotide biosynthesis enzymes are regulated by the phage-induced translational repressor regA, while the T4 single-stranded DNA-binding protein (T4 gp32)...
Autogenous regulatory site on the bacteriophage T4 gene 32 messenger RNA
Journal of Molecular Biology, 1988
We have identified the-binding site on the bacteriophage T4 gene 32 mRNA responsible for autogenous translational regulation. We demonstrate that this site is largely unstructured and overlaps the initiation codon of gene 32 as previously predicted. Cooperative binding of gene 32 protein to this site specifically blocks the formation of 30 S-tRNAy-gene 32 mRNA ternary complexes and initiation of translation. The translational operator is bound cooperatively by gene 32 protein and this binding is facilitated by a nucleation site far upstream from the initiation codon. A similar unstructured mRNA lacking this nucleation site is also bound cooperatively , but only at concentrations of gene 32 protein higher than those needed to repress binding of ribosomes to the gene 32 mRNA. Some sequence-specific interactions may also influence this binding. Comparison of the bacteriophage T2, T4 and T6 gene 32 operator sequences leads us to propose that the nucleation site is a pseudoknot.
Translational regulation of expression of the bacteriophage T4 lysozyme gene. Nucleic Acids Res
1986
The bacteriophage T4 lysozyme gene is transcribed at early and late times after infection of E. coli, but the early mRNA is not translated. DNA sequence analysis and mapping of the 5 ' ends of the lysozyme transcripts produced at different times after T4 infection show that the early mRNA is Initiated some distance upstream from the gene. The early mRNA is not translated because of a stable secondary structure which blocks the translational initiation site. The stable RNA structure has been demonstrated by nudease protection in vivo. After DNA replication begins, two late promoters are activated; the late transcripts are initiated at sites such that the secondary structure can not form, and translation of the late messages occurs.
The Journal of biological chemistry, 1992
The bacteriophage T4 regA protein (M(r) = 14,6000) is a translational repressor of a group of T4 early mRNAs. To identify a domain of regA protein that is involved in nucleic acid binding, ultraviolet light was used to photochemically cross-link regA protein to [32P]p(dT)16. The cross-linked complex was subsequently digested with trypsin, and peptides were purified using anion exchange high performance liquid chromatography. Two tryptic peptides cross-linked to [32P]p(dT)16 were isolated. Gas-phase sequencing of the major cross-linked peptide yielded the following sequence: VISXKQKHEWK, which corresponds to residues 103-113 of regA protein. Phenylalanine 106 was identified as the site of cross-linking, thus placing this residue at the interface of the regA protein-p(dT)16 complex. The minor cross-linked peptide corresponded to residues 31-41, and the site of cross-linking in the peptide was tentatively assigned to Cys-36. The nucleic acid binding domain of regA protein was further e...