Fast recycling of Escherichia coli ribosomes requires both ribosome recycling factor (RRF) and release factor RF3

A complete translation system has been assembled from pure initiation, elongation and termination factors as well as pure aminoacyl‐tRNA synthetases. In this system, ribosomes perform repeated rounds of translation of short synthetic mRNAs which allows the time per translational round (the recycling time) to be measured. The system has been used to study the influence of release factor RF3 and of ribosome recycling factor RRF on the rate of recycling of ribosomes. In the absence of both RF3 and RRF, the recycling time is ∼40 s. This time is reduced to ∼30 s by the addition of RF3 alone and to ∼15 s by the addition of RRF alone. When both RF3 and RRF are added to the translation system, the recycling time drops to <6 s. Release factor RF3 is seen to promote RF1 cycling between different ribosomes. The action of RRF is shown to depend on the concentration of elongation factor‐G. Even in the presence of RRF, ribosomes do not leave the mRNA after termination, but translate the same mRNA several times. This shows that RRF does not actively eject mRNA from the terminating ribosome. It is proposed that terminating ribosomes become mobile on mRNA and ready to enter the next translation round only after two distinct steps, catalysed consecutively by RF3 and RRF, which are slow in the absence of these factors.

[1]  C. Gualerzi,et al.  Selection of the mRNA translation initiation region by Escherichia coli ribosomes. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[2]  L. Mora,et al.  Localization and characterization of the gene encoding release factor RF3 in Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Y. Nakamura,et al.  Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Capecchi,et al.  Polypetide chain termination. Purification of the release factors, R1 and R2, from Escherichia coli. , 1971, The Journal of biological chemistry.

[5]  M. Ehrenberg,et al.  Rate of translation of natural mRNAs in an optimized in vitro system. , 1996, Archives of biochemistry and biophysics.

[6]  A. Kaji,et al.  Reinitiation of translation from the triplet next to the amber termination codon in the absence of ribosome-releasing factor. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[7]  C. Thomas Caskey,et al.  Translational frameshifting: Where will it stop? , 1987, Cell.

[8]  R. Haselkorn,et al.  Studies on the binding of turnip yellow mosaic virus RNA to Escherichia coli ribosomes , 1967 .

[9]  C. Kurland,et al.  The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli. , 1994, Journal of molecular biology.

[10]  P. Jelenc,et al.  Rapid purification of highly active ribosomes from Escherichia coli. , 1980, Analytical biochemistry.

[11]  A. Soffientini,et al.  Purification procedure for bacterial translational initiation factors IF2 and IF3. , 1994, Protein expression and purification.

[12]  A. Hinnebusch,et al.  Requirements for intercistronic distance and level of eukaryotic initiation factor 2 activity in reinitiation on GCN4 mRNA vary with the downstream cistron , 1994, Molecular and cellular biology.

[13]  L. Isaksson,et al.  Emerging Understanding of Translation Termination , 1996, Cell.

[14]  A. Kaji,et al.  Ribosome recycling factor (ribosome releasing factor) is essential for bacterial growth. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  M. Grunberg‐Manago,et al.  Translational regulation of the Escherichia coli threonyl-tRNA synthetase gene: structural and functional importance of the thrS operator domains. , 1993, Biochimie.

[16]  M. Ehrenberg,et al.  Dissociation rates of peptidyl‐tRNA from the P‐site of E.coli ribosomes. , 1996, The EMBO journal.

[17]  C. Caskey,et al.  Peptide chain termination: effect of protein S on ribosomal binding of release factors. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[18]  T. Alatossava,et al.  Manipulation of intracellular magnesium content in polymyxin B nonapeptide-sensitized Escherichia coli by ionophore A23187 , 1985, Journal of bacteriology.

[19]  W P Tate,et al.  Three, four or more: the translational stop signal at length , 1996, Molecular microbiology.

[20]  G. Spedding.  Ribosomes and protein synthesis : a practical approach , 1990 .

[21]  A. Haenni,et al.  A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor , 1994, Nature.

[22]  A. Kaji,et al.  Role of elongation factor G and a protein factor on the release of ribosomes from messenger ribonucleic acid. , 1973, The Journal of biological chemistry.

[23]  H. Bremer.  Modulation of Chemical Composition and Other Parameters of the Cell by Growth Rate , 1999 .

[24]  A. Kaji,et al.  Factor dependent breakdown of polysomes. , 1970, Biochemical and biophysical research communications.

[25]  E. Scolnick,et al.  Peptide chain termination. VI. Purification and site of action of S. , 1970, Proceedings of the National Academy of Sciences of the United States of America.

[26]  C. Kurland,et al.  Nucleoside triphosphate regeneration decreases the frequency of translation errors. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[27]  E. Scolnick,et al.  Peptide chain termination. 3. Stimulation of in vitro termination. , 1969, Proceedings of the National Academy of Sciences of the United States of America.

[28]  A. Hinnebusch,et al.  Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control , 1994, Molecular and cellular biology.

[29]  Chris M. Brown,et al.  Translational termination: "stop" for protein synthesis or "pause" for regulation of gene expression. , 1992, Biochemistry.

[30]  J. van Duin,et al.  Scanning model for translational reinitiation in eubacteria. , 1990, Journal of molecular biology.

[31]  F. Neidhart.  Escherichia coli and Salmonella. , 1996 .

[32]  L. Kisselev,et al.  Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3. , 1995, The EMBO journal.

[33]  A. Kaji,et al.  Ribosome recycling by ribosome recycling factor (RRF)--an important but overlooked step of protein biosynthesis. , 1996, Advances in biophysics.

[34]  M. Ehrenberg,et al.  Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP‐dependent manner , 1997, The EMBO journal.