Abstract
Replacing a cassette of 31 residues from Escherichia coli release factor 1 with the equivalent residues in release factor 2 gave a protein active in codon-specific binding to the ribosome but inactive in peptidyl-tRNA hydrolysis. Such a phenotype is also found unexpectedly with release factor 2 when expressed at high concentration in bacteria. Substituting threonine with the release factor 1 equivalent serine at position 246 within the cassette restored the impaired activity of the chimeric protein, and also that of inactive recombinant release factor 2, both in vitro and in vivo. The differences in activity are not due to posttranslational modifications or a lack of it at this residue. Random mutagenesis of codon 246 suggests that this position is pivotal for the function of the release factor, being able to affect differentially both its binding to the ribosome and its peptide release activities. We propose that amino acid 246 is close to a sharp turn (GGQ motif at position 250), and is essential for transmitting the signal from cognate codon recognition by correctly positioning the peptidyl-tRNA hydrolysis domain of the release factor into the peptidyltransferase center.
Full Text
The Full Text of this article is available as a PDF (614.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Arkov A. L., Freistroffer D. V., Ehrenberg M., Murgola E. J. Mutations in RNAs of both ribosomal subunits cause defects in translation termination. EMBO J. 1998 Mar 2;17(5):1507–1514. doi: 10.1093/emboj/17.5.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brock S., Szkaradkiewicz K., Sprinzl M. Initiation factors of protein biosynthesis in bacteria and their structural relationship to elongation and termination factors. Mol Microbiol. 1998 Jul;29(2):409–417. doi: 10.1046/j.1365-2958.1998.00893.x. [DOI] [PubMed] [Google Scholar]
- Brown C. M., McCaughan K. K., Tate W. P. Two regions of the Escherichia coli 16S ribosomal RNA are important for decoding stop signals in polypeptide chain termination. Nucleic Acids Res. 1993 May 11;21(9):2109–2115. doi: 10.1093/nar/21.9.2109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown C. M., Tate W. P. Direct recognition of mRNA stop signals by Escherichia coli polypeptide chain release factor two. J Biol Chem. 1994 Dec 30;269(52):33164–33170. [PubMed] [Google Scholar]
- Capecchi M. R., Klein H. A. Characterization of three proteins involved in polypeptide chain termination. Cold Spring Harb Symp Quant Biol. 1969;34:469–477. doi: 10.1101/sqb.1969.034.01.053. [DOI] [PubMed] [Google Scholar]
- Caskey T., Scolnick E., Tompkins R., Goldstein J., Milman G. Peptide chain termination, codon, protein factor, and ribosomal requirements. Cold Spring Harb Symp Quant Biol. 1969;34:479–488. doi: 10.1101/sqb.1969.034.01.054. [DOI] [PubMed] [Google Scholar]
- Crawford D. J., Ito K., Nakamura Y., Tate W. P. Indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli release factor RF3. EMBO J. 1999 Feb 1;18(3):727–732. doi: 10.1093/emboj/18.3.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Donly B. C., Edgar C. D., Adamski F. M., Tate W. P. Frameshift autoregulation in the gene for Escherichia coli release factor 2: partly functional mutants result in frameshift enhancement. Nucleic Acids Res. 1990 Nov 25;18(22):6517–6522. doi: 10.1093/nar/18.22.6517. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frolova L. Y., Tsivkovskii R. Y., Sivolobova G. F., Oparina N. Y., Serpinsky O. I., Blinov V. M., Tatkov S. I., Kisselev L. L. Mutations in the highly conserved GGQ motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis. RNA. 1999 Aug;5(8):1014–1020. doi: 10.1017/s135583829999043x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito K., Uno M., Nakamura Y. A tripeptide 'anticodon' deciphers stop codons in messenger RNA. Nature. 2000 Feb 10;403(6770):680–684. doi: 10.1038/35001115. [DOI] [PubMed] [Google Scholar]
- McCaughan K. K., Poole E. S., Pel H. J., Mansell J. B., Mannering S. A., Tate W. P. Efficient in vitro translational termination in Escherichia coli is constrained by the orientations of the release factor, stop signal and peptidyl-tRNA within the termination complex. Biol Chem. 1998 Jul;379(7):857–866. doi: 10.1515/bchm.1998.379.7.857. [DOI] [PubMed] [Google Scholar]
- Mikuni O., Kawakami K., Nakamura Y. Sequence and functional analysis of mutations in the gene encoding peptide-chain-release factor 2 of Escherichia coli. Biochimie. 1991 Dec;73(12):1509–1516. doi: 10.1016/0300-9084(91)90185-4. [DOI] [PubMed] [Google Scholar]
- Moazed D., Samaha R. R., Gualerzi C., Noller H. F. Specific protection of 16 S rRNA by translational initiation factors. J Mol Biol. 1995 Apr 28;248(2):207–210. doi: 10.1016/s0022-2836(95)80042-5. [DOI] [PubMed] [Google Scholar]
- Moffat J. G., Donly B. C., McCaughan K. K., Tate W. P. Functional domains in the Escherichia coli release factors. Activities of hybrids between RF-1 and RF-2. Eur J Biochem. 1993 Apr 15;213(2):749–756. doi: 10.1111/j.1432-1033.1993.tb17816.x. [DOI] [PubMed] [Google Scholar]
- Moffat J. G., Tate W. P. A single proteolytic cleavage in release factor 2 stabilizes ribosome binding and abolishes peptidyl-tRNA hydrolysis activity. J Biol Chem. 1994 Jul 22;269(29):18899–18903. [PubMed] [Google Scholar]
- Nakamura Y., Ito K. How protein reads the stop codon and terminates translation. Genes Cells. 1998 May;3(5):265–278. doi: 10.1046/j.1365-2443.1998.00191.x. [DOI] [PubMed] [Google Scholar]
- Nissen P., Kjeldgaard M., Nyborg J. Macromolecular mimicry. EMBO J. 2000 Feb 15;19(4):489–495. doi: 10.1093/emboj/19.4.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pavlov M. Y., Freistroffer D. V., Dincbas V., MacDougall J., Buckingham R. H., Ehrenberg M. A direct estimation of the context effect on the efficiency of termination. J Mol Biol. 1998 Dec 4;284(3):579–590. doi: 10.1006/jmbi.1998.2220. [DOI] [PubMed] [Google Scholar]
- Pel H. J., Rep M., Grivell L. A. Sequence comparison of new prokaryotic and mitochondrial members of the polypeptide chain release factor family predicts a five-domain model for release factor structure. Nucleic Acids Res. 1992 Sep 11;20(17):4423–4428. doi: 10.1093/nar/20.17.4423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Poole E. S., Brown C. M., Tate W. P. The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J. 1995 Jan 3;14(1):151–158. doi: 10.1002/j.1460-2075.1995.tb06985.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Song H., Mugnier P., Das A. K., Webb H. M., Evans D. R., Tuite M. F., Hemmings B. A., Barford D. The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell. 2000 Feb 4;100(3):311–321. doi: 10.1016/s0092-8674(00)80667-4. [DOI] [PubMed] [Google Scholar]
- Uno M., Ito K., Nakamura Y. Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2. Biochimie. 1996;78(11-12):935–943. doi: 10.1016/s0300-9084(97)86715-6. [DOI] [PubMed] [Google Scholar]