Fusions of bacteriophage P22 late genes to the Escherichia coli lacZ gene (original) (raw)
Related papers
Regulation of bacteriophage P2 late-gene expression: the ogr gene
Proceedings of the National Academy of Sciences of the United States of America, 1986
The ogr gene product of bacteriophage P2 is a positive regulatory factor required for P2 late-gene transcription. We have determined the nucleotide sequence of the ogr gene, which encodes a basic polypeptide of 72 amino acids. P2 growth is blocked by a host mutation, rpoA09, in the a subunit of DNA-dependent RNA polymerase. The ogr52 mutation, which allows P2 to grow in an rpoAl09 strain, was shown to be a single nucleotide change, in the codon for residue 42, that changes tyrosine to cysteine. The predicted amino acid sequence of the Ogr protein does not show similarity to DNAbinding proteins that are known to affect promoter recognition, to a factors, or to other characterized transcriptional regulatory proteins. We have inserted the ogr gene into a plasmid under control of the leftward promoter and operator of bacteriophage X. Thermal induction of ogr gene expression in this plasmid results in overproduction of a small protein that has been shown by complementation to possess Ogr function.
Translational signals of a major head protein gene of bacteriophage lambda
MGG Molecular & General Genetics, 1988
The D gene of bacteriophage 2 which codes for a major head protein is expressed at a high level during lytic growth. We have constructed a set of D-lacZ gene fusions in order to examine the factors determining the high efficiency of the D translational initiation signals. It was found that an integral sequence, 300 bp long and upstream of the ATG initiation codon, is required for maximal protein synthesis.
Identification of Upstream Sequences Essential for Activation of a Bacteriophage P2 Late Promoter
Journal of Bacteriology, 2003
We have carried out a mutational scan of the upstream region of the bacteriophage P2 FETUD late operon promoter, P F , which spans an element of hyphenated dyad symmetry that is conserved among all six of the P2 and P4 late promoters. All mutants were assayed for activation by P4 Delta in vivo, by using a lacZ reporter plasmid, and a subset of mutants was assayed in vitro for Delta binding. The results confirm the critical role of the three complementary nucleotides in each half site of the upstream element for transcription factor binding and for activation of transcription. A trinucleotide DNA recognition site is consistent with a model in which these transcription factors bind via a zinc finger motif. The mutational scan also led to identification of the ؊35 region of the promoter. Introduction of a 70 ؊35 consensus sequence resulted in increased constitutive expression, which could be further stimulated by Delta. These results indicate that activator binding to the upstream region of P2 late promoters compensates in part for poor 70 contacts and helps to recruit RNA polymerase holoenzyme.
Proceedings of the National Academy of Sciences, 1988
The late operon of bacteriophage A contains the genes encoding the morphogenetic proteins of the phage. These genes are transcribed equally from the single late promoter. Although the functional half-lives of the mRNA for the various genes of this operon vary <2-fold, their relative rates of expression have been shown to vary by nearly 1000fold. This variation could result from differing rates of translation initiation, from overlapping upstream translation, or from differential elongation rates due to the presence of codons for which the corresponding tRNAs are rare. To distinguish between these possibilities, we have cloned sequences surrounding the initiator codons of several of these genes and measured their ability to drive synthesis of hybrid A-agalactosidase proteins. The rates of expression of the hybrid genes thus produced correlate very well with the natural rates of expression of the corresponding phage genes, suggesting that the rate of initiation of translation controls the relative expression rates of these genes.
Journal of Biological Chemistry
We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S . (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 11.0-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as: a translational "enhancer."
Translational control of the expression of bacteriophage T7 gene 0.3
Journal of Molecular Biology, 1978
When Escherichia coli are infected at 43°C with a bacteriophage T7 mutant that produces a temperature-sensitive RNA polymerase (ts342), the rate of transcription of the T7 late genes is reduced three-to fourfold below the rate of transcription in cells infected with wild-type T7. The reduction in T7 late mRNA concentration in cells infected with ts342 is accompanied by the overproduction at late times of at least one T7 early protein, the gene O-3 protein. Despite the difference in T7 late mRNA concentration in cells infected with wild-type T7 and ts342 at 43"C, T7 late proteins are synthesized at the same rate in the two infectedcell cultures. These findings support an hypothesis of discrimination against 0.3 mRNA translation in favor of translating T7 late messages when mRNA is in excess of the protein synthetic machinery of the cell.
Deletion analysis of a bacteriophage P2 late promoter
Gene, 1990
We have fused the promoter (PF) for the P2 late FETUD oceron to the gene (cat) encoding chloramphenicol acetyltransferase (CAT) in a plasmid vector. Synthesis of CAT in Escherichia coli strains carrying this plasmid requires the product of the P2 ogr gene or the satellite phage P4 transactivation gene, 6. Our results demonstrate that these phage-encoded transcriptional regulatory proteins are necessary and sufficient for activation of P2 late transcription in this reporter plasmid. Positive regulation of cloned PF is severely impaired in a host strain carrying the rpod 109 mutation. Expression from the cloned promoter thus approximates those features of P2 late transcription that have been shown to occur during normal P2 infection. To define sequences required for promoter function, sequential upstream deletions of Pv were generated using BAL 31 nuclease, and the mutant promoters were assayed for cat expression. A sequence between nucleotides-69 and-64 from the transcription start point was found to be essential for promoter activity. This coincides with a region of homology conserved among all four P2 late gene promoters and the two P4 late promoters, and includes an element of dyad symmetry.
Journal of Molecular Biology, 1989
Two plasmid systems, containing the easily assayable galK and 1acZ functions, were employed to study the regulation of the bacteriophage Pl tail-fibre and dar operons. Various PI DNA fragments carrying either the 5' end of 2ydA (the 1st gene in the dar operon) or the tail-fibre gene 19 precede the promoterless coding region of galK or were fused, in-frame, to the 1acZ gene. In the presence of an induced Pl prophage, GalK and LacZ activities were both detected after a 20 to 30 minute lag period, indicating that the dar and tail-fibre operons are expressed from positively regulated, late promoters. The corresponding DNA region of the closely related p15B plasmid exhibits comparable promoter properties. Deletion analysis mapped the promoter of a gene 19-&Z fusion to a DNA region upstream from gene R, an open reading frame that precedes the coding frame of gene 19. The tail-fibre gene thus forms the second gene in a three gene operon (genes R, 19 (S) and U). Sequence comparison between this promoter region, upstream sequences of the 1ydA gene and the corresponding portions of the p15B genome allowed the identification of a highly conserved 38 base-pair sequence, which most likely represents a Pl-specific late promoter. This was confirmed by 5' mapping of Pl mRNA. Transcription of both the tail-fibre and dar operons is initiated at sites five and six base-pairs, respectively, downstream from the first conserved nucleotide of this sequence. The conserved motif consists of a standard Escherichia coli -10 region followed by a nine base-pair palindromic sequence located centrally about position -22.
Nucleic Acids Research, 1978
We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediatly downstream from the lac UV5promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), whi'ch shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison g6netique in vitro" committee (D.G.R.S.T.).
Journal of Bacteriology, 1985
We have constructed several derivatives of bacteriophage A that translocate by using the transposition machinery of phage Mu (A pldcMu phages). Each phage carries the c end of Mu, containing the Mu cIts62, ner (cII), and A genes, and the terminal sequences from the Mu S end (,B end). These sequences contain the Mu attachment sites, and their orientation allows the A genome to be inserted into other chromosomes, resulting in a K prophage flanked by the Mu c and S sequences. These phages provide a means to isolate cells containing fusions of the lac operon to other genes in vivo in a single step. In k placMu50, the lacZ and lacY genes, lacking a promoter, were located adjacent to the Mu S sequence. Insertion of A placMu5O into a gene in the proper orientation created an operon fusion in which lacZ and lacY were expressed from the promoter of the target gene. We also introduced a gene, kan, which confers kanamycin resistance, into A placMuS0 and A placMul, an analogous phage for constructing lacZ protein fusions (Bremer et al., J. Bacteriol. 158:1084-1093, 1984). The kan gene, located between the cIII and ssb genes of A, permitted cells containing insertions of these phages to be selected independently of their Lac phenotype.