Cloning, nucleotide sequence, and overexpression of the bacteriophage T4 regA gene (original) (raw)

The bacteriophage T4 regA gene: primary sequence of a translational repressor

Nucleic Acids Research, 1984

The regA gene product of bacteriophage T4 is an autogenously controlled translational regulatory protein that plays a role in differential inhibition (translational repression) of a subpopulation of T4-encoded "early" mRNA species. The structural gene for this polypeptide maps within a cluster of phage DNA replication genes, (genes 45-44-62-regA-43-42), all but one of which (gene 43) are under regA-mediated translational control. We have cloned the T4 regA gene, determine its nucleotide sequence, and identified the amino-terminal residues of a plasmid-encoded, hyperproduced regA protein. The results suggest that the T4 regA gene product is a 122 amino acid polypeptide that is mildly basic and hydrophilic in character; these features are consistent with known properties of regA protein derived from T4-infected cells. Computer-assisted analyses of e nucleotide sequences of the regA gene and its three upstream neighbors (genes 45, 44, and 62) suggest tWe existence of three translational initiation units in this four-gene cluster; one for gene 45, one for genes 44, 62 and regA, and one that serves only the egA gene. The analyses also suggest that the gene 44-62 translational unit harbors a stable RNA structure that obligates translational coupling of these two genes.

Bacteriophage T4, a new vector for the expression of cloned genes

Gene, 1985

The amino-terminal portion of the T4 rIIB gene has been fused to the coding sequence of a truncated ZacZ gene from Escherichiu co& giving rise to a fusion protein with fi-galactosidase activity. The 3 192-bp rIIB-ZacZ gene fusion was transferred into phage T4, and enzymatically active protein was produced after phage infection. T4 may be a useful expression vector in special circumstances, in particular for proteins whose accumulation in E. cob is limited by sensitivity to proteases.

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

Journal of Molecular Biology, 1981

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 300/b of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gpll , before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 D?r'A have @loi, and mini cells have lTO of the gpll levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gpl0 and gpll formed from the cloned genes after T4 infection.

Identification of amino acid residues at the interface of a bacteriophage T4 regA protein-nucleic acid complex

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...

Gene regulation at the right operator (OR) of bacteriophage l

1980

In a X lysogen, transcription of the repressor gene, c1, initiates at the promoter P,, (see, for example, Reichardt & Kaiser, 197 1). Construction of a hybrid operon is described in which this promoter directs transcription of the trpA and la& genes permitting facile analysis of the regulation of P,, in viva. As reported previously , low levels of repressor stimulate P,, while higher levels of repressor turn it off. We report that mutations which render P,, insensitive to turnoff by repressor are found in Oa3, an operator site which overlaps P,,.

DNA sequences of the repressor gene and operator region of bacteriophage P2

Proceedings of the National Academy of Sciences, 1984

The nucleotide sequence of the repressor gene C of the temperate phage P2 has been determined. It codes for a nonbasic polypeptide, 99 amino acids long. Twelve repressor-defective mutants have been mapped. All but one are located within the presumed coding part of the gene. There is a strong promoter sequence and an 8-base-pair inverted repeat preceding the gene. The P2 repressor protein shows structural similarity to other DNA-binding proteins. The operator region for the early replication functions was located by sequencing the DNA of three virulent mutants. The sequence indicates that there are two repressor-binding sites. In addition, one of the sites shows sequence homology with part of the operator region of the biotin operon of Escherichia coli.

Nucleotide sequence and complementation studies of the gene 10 region of bacteriophage T3

Journal of Molecular Biology, 1989

The nucleotide sequence of bacteriophage T3 gene 10 and surrounding regulatory elements has been determined and compared to the analogous region of bacteriophage T7. T3 genes 9, 10 and 11 have been shown to complement T7 mutants. The DNA sequences of T3 and T7 gene IOA are homologous, as are the amino acid sequences of the respective products. The translational shift to the-1 frame is predicted to occur at the same position in gene 10 of T3 and T7, though different nucleotide sequences are probably responsible. The resulting gplOB products have completely different C termini.