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.

[1]  Guy Plunkett,et al.  Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript , 1989, Journal of bacteriology.

[2]  U. Brinkmann,et al.  High-level expression of recombinant genes in Escherichia coli is dependent on the availability of the dnaY gene product. , 1989, Gene.

[3]  P. Reeves,et al.  Intermediates in the synthesis of TolC protein include an incomplete peptide stalled at a rare Arg codon. , 1985, European journal of biochemistry.

[4]  V. Emilsson,et al.  Growth-rate-dependent accumulation of twelve tRNA species in Escherichia coli. , 1993, Journal of molecular biology.

[5]  D. Court,et al.  Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[6]  D. Schindler,et al.  Retroregulation of the int gene of bacteriophage lambda: control of translation completion. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[7]  S. Adhya,et al.  Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda , 1976, Journal of bacteriology.

[8]  S. Rangwala,et al.  A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. , 1989, The Journal of biological chemistry.

[9]  C. Kurland,et al.  Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction , 1995, Journal of bacteriology.

[10]  K. Jensen,et al.  The AGG codon is translated slowly in E. coli even at very low expression levels. , 1988, Nucleic acids research.

[11]  W. Gilbert,et al.  Sequencing end-labeled DNA with base-specific chemical cleavages. , 1980, Methods in enzymology.

[12]  Guy Plunkett,et al.  DNA sequence of the transfer RNA region of bacteriophage T4: implications for transfer RNA synthesis. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[13]  H. Nash,et al.  Nicking-closing activity associated with bacteriophage lambda int gene product. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[14]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[15]  J. Walker,et al.  Expression of argU, the Escherichia coli gene coding for a rare arginine tRNA , 1992, Journal of bacteriology.

[16]  M. Inouye,et al.  Suppression of the negative effect of minor arginine codons on gene expression; preferential usage of minor codons within the first 25 codons of the Escherichia coli genes. , 1990, Nucleic acids research.

[17]  J. Walker,et al.  A minor arginine tRNA mutant limits translation preferentially of a protein dependent on the cognate codon , 1990, Journal of bacteriology.

[18]  L. Matsumoto,et al.  Site-specific recombination intermediates trapped with suicide substrates , 1987, Cell.

[19]  M. Rosenberg,et al.  Purified λ regulatory protein cII positively activates promoters for lysogenic development , 1981, Nature.

[20]  S. Gottesman,et al.  Bacteriophage λ: The Lysogenic Pathway , 1977 .

[21]  C. Kurland,et al.  Codon usage determines translation rate in Escherichia coli. , 1989, Journal of molecular biology.

[22]  Stanley N Cohen,et al.  Genetic expression in bacteriophage λ: III. Inhibition of Escherichia coli nucleic acid and protein synthesis during λ development , 1970 .

[23]  J. van Duin,et al.  Translation of the sequence AGG-AGG yields 50% ribosomal frameshift. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Murray,et al.  Sequence diversity among related genes for recognition of specific targets in DNA molecules. , 1983, Journal of molecular biology.

[25]  B. Oakley,et al.  A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. , 1980, Analytical biochemistry.

[26]  J R Lorsch,et al.  Reverse transcriptase reads through a 2'-5'linkage and a 2'-thiophosphate in a template. , 1995, Nucleic acids research.

[27]  T. Ikemura.  Correlation between the abundance of Escherichia coli transfer RNAs and the occurrence of the respective codons in its protein genes: a proposal for a synonymous codon choice that is optimal for the E. coli translational system. , 1981, Journal of molecular biology.