Int Protein Synthesis Research Articles

The pair of arginine codons AGA AGG close to the initiation codon of the lambda int gene inhibits cell growth and protein synthesis by accumulating peptidyl-tRNAArg4.

To analyse the mechanism by which rare codons near the initiation codon inhibit cell growth and protein synthesis, we used the bacteriophage lambda int gene or early codon substitution derivatives. The lambda int gene has a high frequency of rare ATA, AGA and AGG codons; two of them (AGA AGG) located at positions 3 and 4 of the int open reading frame (ORF). Escherichia coli pth (rap) cells, which are defective in peptidyl-tRNA hydrolase (Pth) activity, are more susceptible to the inhibitory effects of int expression as compared with wild-type cells. Cell growth and Int protein synthesis were enhanced by overexpression of Pth and tRNAArg4 cognate to AGG and AGA but not of tRNAIle2a specific for ATA. The increase of Int protein synthesis also takes place when the rare arginine codons AGA and AGG at positions 3 and 4 are changed to common arginine CGT or lysine AAA codons but not to rare isoleucine ATA codons. In addition, overexpression of int in Pth defective cells provokes accumulation of peptidyl-tRNAArg4 in the soluble fraction. Therefore, cell growth and Int synthesis inhibition may be due to ribosome stalling and premature release of peptidyl-tRNAArg4 from the ribosome at the rare arginine codons of the first tandem, which leads to cell starvation for the specific tRNA.

Overexpression of an mRNA dependent on rare codons inhibits protein synthesis and cell growth.

lambda's int gene contains an unusually high frequency of the rare arginine codons AGA and AGG, as well as dual rare Arg codons at three positions. Related work has demonstrated that Int protein expression depends on the rare AGA tRNA. Strong transcription of the int mRNA with a highly efficient ribosome-binding site leads to inhibition of Int protein synthesis, alteration of the overall pattern of cellular protein synthesis, and cell death. Synthesis or stability of int and ampicillin resistance mRNAs is not affected, although a portion of the untranslated int mRNA appears to be modified in a site-specific fashion. These phenotypes are not due to a toxic effect of the int gene product and can be largely reversed by supplementation of the AGA tRNA in cells which bear plasmids expressing the T4 AGA tRNA gene. This indicates that depletion of the rare Arg tRNA due to ribosome stalling at multiple AGA and AGG codons on the overexpressed int mRNA underlies all of these phenomena. It is hypothesized that int mRNA's effects on protein synthesis and cell viability relate to phenomena involved in lambda phage induction and excision.

Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript

Expression of the int gene of bacteriophage lambda from two promoters, pI and pL, is differentially regulated through RNA processing. Efficient Int protein synthesis from the pL RNA is inhibited by the action of sib, a cis-acting retroregulator downstream from the int gene. We have used mapping procedures with nuclease S1 to study the pL transcripts produced in vivo after phage lambda infection. We have found an RNase III-dependent processing site within the Int coding sequence, 387 nucleotides upstream from the site of the primary cleavage by RNase III at Sib. This secondary processing site is located at the most stable region of secondary structure in the sib int region, as predicted by computer analysis. We suggest that RNase III cleavage at the Sib site allows processive exonucleolytic degradation of the RNA to proceed to a region of secondary structure within the Int coding sequence, which protects the upstream region of the transcript from further degradation.

Transcription terminator involved in the expression of the int gene of phage lambda

The phage λ int gene is transcribed from two different promoters, p i and p l . Transcription from p i results in efficient synthesis of Int protein whereas transcription originating from p l results in poor int expression. The differential expression of Int from these two transcripts is dependent upon a site ( sib) located distal to the int gene [Guameros and Galindo, Virology 95 (1979) 119–126; Guameros et al., Proc. Natl. Acad. Sci. USA 79 (1982) 238–242]. We have examined p i -promoted transcription in the region beyond the int coding sequence. The int mRNA extends to a site designated t i , which is located 277 nucleotides beyond int. Characterization of transcription at t i indicates that t i terminates with 75% efficiency in vitro, and that its efficiency is over 95% in vivo. The region between int and t i contains the regulatory signals needed for phage λ integration and appears to be untranslated. The termination site overlaps with the sib control region that reduces Int synthesis from p l transcripts.

Multilevel regulation of bacteriophage λ lysogeny by the E. coli himA gene

Previous experiments have shown that the himA gene of E. coli specifies a protein that is required for bacteriophage λ integration. λ forms clear plaques on himA mutants indicating a possible additional defect in the establishment of repression. We have tested the effects of a himA mutation on the establishment and maintenance of λ repressor (cl) synthesis and on the synthesis of Int protein. The rate of synthesis of cl and Int after infection by λ is severely reduced in a strain carrying a himA gene deletion. Synthesis of Int or repressor can occur in the himA − strain if the phage carry constitutive promoters for either the int gene or the cl gene. Maintenance of repression is unaffected by himA mutations as judged by repressor-stimulated transcription of P M fused to the lacZ gene. These results indicate that the himA gene participates in the regulation of the promoter sites specific for the establishment of lysogeny: P E for cl synthesis and P E for Int production. Since the himA gene product is required also for λ site-specific recombination, it appears that the himA gene regulates λ lysogeny at several levels. I discuss the significance of this multilevel regulation to λ development.

Retroregulation of the int gene of bacteriophage lambda: control of translation completion.

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.

Downstream regulation of int gene expression by the b2 region in phage lambda

Expression of the int gene after phage λ infection normally requires the products of genes cII and cIII. However, when the phage carries a deletion in the nonessential b2 region adjacent to int, efficient synthesis of active Int protein does not require cII and cIII function. This inhibition of Int synthesis by nucleotide sequences downstream from the int structural gene behaves in a cis-dominant fashion in mixed infections. It is specific for P L- and not p I-initiated transcripts. Based on these observations, and those of others, a model is proposed in which Int translation from the p L transcript is inhibited by the interaction of downstream b2 nucleotide sequences and nucleotide sequences in the int region. The data imply a novel temporal mechanism regulating prophage λ induction: circularization of the prophage genome results in the transposition of inhibitory b2 region sequences next to int and blocks further Int protein synthesis beyond the low level required for excision. As a consequence of this process, the control of int expression is transferred from the p L promoter to p I and the cII/ cIII system. Such a genetic regulatory mechanism involving the rearrangement of genetic elements downstream from a structural gene may be of general use during development in other systems.

Functional analysis of hybrid plasmids carrying genes for λ site-specific recombination

A number of hybrid plasmids, carrying λ genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::λ-CD, RSF2124::λ-C int-c57, and pIntλ were able to integrate into the chromosome of E. coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [ 32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pIntλ demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of λ are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pIntλ 2 as compared to pIntλ 1) is critical both for integration and excision.

Transcription of the int gene of bacteriophage λ new RNA polymerase binding site and RNA start generated by int-constitutive mutations

High level synthesis of the λ Int protein depends on positive regulation by the λ cII/cIII proteins, which enhance transcription from p I, a promoter close to the int gene. The intc point mutations allow high level synthesis of the Int protein even when the cII/cIII proteins are absent. In order to understand how the intc point mutations increase the constitutive level of Int protein synthesis, we have carried out a series of binding and transcription experiments in vitro using purified Escherichia coli RNA polymerase. We have studied the binding of RNA polymerase to a DNA restriction fragment that contains the p I promoter region; we find that RNA polymerase has a much greater affinity for this restriction fragment if it has an intc point mutation. We have also transcribed the restriction fragment in vitro and analyzed the RNA products on agarose/polyacrylamide gels. If the DNA has an intc point mutation, a leftward transcript of the int gene is detected; no leftward transcripts are detected when the DNA template does not have an intc point mutation. From these results we conclude that the intc point mutations have generated an active promoter site for int gene transcription.