Genetic analysis of bacteriophage λcIII gene: mRNA structural requirements for translation initiation (original) (raw)
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RNase III stimulates the translation of the cIII gene of bacteriophage lambda
Proceedings of the National Academy of Sciences, 1987
The bacteriophage X cMII gene product regulates the lysogenic pathway by stabilizing the X cil regulatory protein. Our results show that the expression of the X cMII gene is subject to specific requirements. Tests of a set of cHl-acZ gene and operon fusions reveal that a sequence upstream of the cd ribosome binding site is needed for cMII translation. The sequence contains an inefficient RNase III processing site. Furthermore, expression of cM is drastically reduced in cells lacking RNase III. We have isolated a phage carrying a mutation (rl), which lies in the upstream sequence, that leads to a reduction in cII translation and inactivates the RNase m processing site. The ri mutant is nevertheless still dependent on RNase III for cMII translation; ri reduces cII1 translation by a factor of 3 in wild-type cells and by a factor of =30 in an RNase III mutant host. We propose that RNase Ill stimulates MI translation by binding to the upstream sequence and thereby exposing the cMII ribosome binding site. This stimulation does not involve RNA cleavage. Consistent with this hypothesis is our finding that, in vitro, unprocessed cII mRNA is translated, whereas RNase III-cleaved cIul mRNA is not.
Genetic analysis of the cIII gene of bacteriophage HK022
Journal of bacteriology, 1991
The cIII gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. Previous works showed that the synthesis of the bacteriophage lambda CIII protein has specific translational requirements imposed by the structure of the mRNA. To gain insight into the mRNA structure and its role in regulating cIII translation, we undertook a mutational analysis of the cIII gene of the related bacteriophage HK022. Our data support the hypothesis that in HK022, as in lambda, translation initiation requires a specific mRNA structure. In addition, we found that translation of HK022 cIII, like that of lambda, is strongly reduced in a host deficient in the endonuclease RNase III.
1990
The clII gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded CII protein, a transcriptional activator of the repressor and integrase genes. Previous works showed that the synthesis of the bacteriophage A CIII protein has specific translational requirements imposed by the structure of the mRNA. To gain insight into the mRNA structure and its role in regulating cIII translation, we undertook a mutational analysis of the cIII gene of the related bacteriophage HK022. Our data support the hypothesis that in HK022, as in X, translation initiation requires a specffic mRNA structure. In addition, we found that translation of HK022 cIII, like that of X, is strongly reduced in a host deficient in the endonuclease RNase III. HK022 is a temperate bacteriophage of Escherichia coli (9). Its ability to form viable recombinants with bacterio-phage A shows that it is a member of the lambdoid phage family (10, 25). The lambdoid phages have a similar organi-zatio...
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.
Translation initiation of bacteriophage lambda gene cII requires integration host factor
Journal of bacteriology, 1986
Escherichia coli integration host factor (IHF), a DNA-binding protein, positively regulates expression of the lambda cII gene. Purified IHF stimulates cII protein synthesis in vitro, suggesting a direct role for host factor in cII expression. Further evidence for a direct role for IHF was obtained with operon and gene fusions between cII and lacZ or cII and galE. Analysis of these fusions in vivo demonstrated that IHF is essential for the initiation of cII translation. Replacement of the entire cII coding sequence with lacZ yielded a gene fusion which was still IHF dependent. However, a cII-galE fusion carrying a hybrid ribosome binding region expressed galE in IHF mutants. These results indicate that sequences which make cII translation IHF dependent lie between the ribosome binding region and the initiating codon of cII. Failure to translate cII activates a transcription terminator located within cII and results in polar effects on downstream transcription. This polarity is suppre...
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.
Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene
Proceedings of the National Academy of Sciences of the United States of America, 1982
The bacteriophage A int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by A proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression ofint from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. A mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240-bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hefmutatsons cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription.of the int gene. There is no difference in the rate of .PL'promoted int mRNA synthesis in either sib' or sib-phage infections, yet int mRNA is less stable in the sib' infection. Because RNase 111 host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.
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.
Translationsl regulation of expression of the bacteriophage T4 lysozyme gene
Nucleic Acids Research, 1986
The bacteriophage T4 lysozyme aene 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 aene. 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 nuclease protection in vivo. After DNA replication begins, two late promoters are activated; thTe late transcripts are initiated at sites such that the secondary structure can not form, and translation of the late messages occurs.
Molecular and General Genetics MGG, 1978
The activation of the int gene by the cII and cIII gene products was studied by analysing int expression following infection of UV-irradiated cells by various phage mutants. Residual expression of int, probably from PL, takes place in the absence of cII/ cIII activation. Activation of the int gene, like that of the cI repressor gene, is poor at low multiplicities of infection. The mutation intC, which allows constitutive int expression in the lysogenic state, partially relieves the requirement for cII and cIII activation. The kinetics of Int synthesis after addition of the inhibitor rifampicin suggest that the activation occurs at the transcriptional level.