Initiation of in vivo protein synthesis with non-methionine amino acids

Chattapadhyay, Ranjan; Pelka, Heike; Schulman, LaDonne H.

doi:10.1021/bi00470a001

Cited by 67 publications

(58 citation statements)

References 32 publications

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“…This could be due to a difference in sequence or due to a difference in base modification in the anticodon loop of the mutant tRNAs (30), as a consequence of the presence of an A rather than a G at position 36. Modification of A37 in the anticodon loop to i 6 A is strongly dependent on the presence of A36 (4), which is present in the U35A36 but not in the U35G36 mutant tRNA.…”

Section: Resultsmentioning

confidence: 99%

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Drabkin

RajBhandary

1998

Molecular and Cellular Biology

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Protein synthesis is initiated universally with the amino acid methionine. In Escherichia coli, studies with anticodon sequence mutants of the initiator methionine tRNA have shown that protein synthesis can be initiated with several other amino acids. In eukaryotic systems, however, a yeast initiator tRNA aminoacylated with isoleucine was found to be inactive in initiation in mammalian cell extracts. This finding raised the question of whether methionine is the only amino acid capable of initiation of protein synthesis in eukaryotes. In this work, we studied the activities, in initiation, of four different anticodon sequence mutants of human initiator tRNA in mammalian COS1 cells, using reporter genes carrying mutations in the initiation codon that are complementary to the tRNA anticodons. The mutant tRNAs used are aminoacylated with glutamine, methionine, and valine. Our results show that in the presence of the corresponding mutant initiator tRNAs, AGG and GUC can initiate protein synthesis in COS1 cells with methionine and valine, respectively. CAG initiates protein synthesis with glutamine but extremely poorly, whereas UAG could not be used to initiate protein synthesis with glutamine. We discuss the potential applications of the mutant initiator tRNA-dependent initiation of protein synthesis with codons other than AUG for studying the many interesting aspects of protein synthesis initiation in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Drabkin

RajBhandary

1998

Molecular and Cellular Biology

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“…Moreover, such initiation of translation on a non-AUG codon does not require that the suppressor tRNA be aminoacylated with methionine (3,19,38). Recently, advantage was taken of these properties to compare in vivo the initiator activity of several mutants of E. coli tRNAfmet harboring a CUA anticodon.…”

Section: Discussionmentioning

confidence: 99%

Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli

et al. 1993

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In bacteria, the free amino group of the methionylated initiator tRNA is specifically modified by the addition of a formyl group. The functional relevance of such a formylation for the initiation of translation is not yet precisely understood. Advantage was taken here of the availability of thefint gene, encoding the Escherichia coli Met-tRNA;Met formyltransferase, to measure the influence of variations in the level of formyltransferase activity on the involvement of various mutant tRNAftet and tRNA:jet species in either initiation or elongation in vivo. The data obtained established that formylation plays a dual role, firstly, by dictating tRNA;Iet to engage in the initiation of translation, and secondly, by preventing the misappropriation of this tRNA by the elongation apparatus. The importance of formylation in the initiator identity of tRNA;Yet was further shown by the demonstration that elongator tRNA~et may be used in initiation and no longer in elongation, provided that it-is mutated into a formylatable species and is given the three G* C base pairs characteristic of the anticodon stem of initiator tRNAs.The recognition of initiator Met-tRNA by specific factors governs the initiation of translation (35). In addition, initiator Met-tRNA must not be recognized by the elongation apparatus, which uses a distinct tRNAMet for the incorporation of internal methionines.In procaryotic cells, prior to its involvement in translation, initiator Met-tRNAr~et is modified by the addition of a formyl moiety on the NH2 group of the esterified methionine. This reaction, catalyzed by 10-formyltetrahydrofolate:L-methionyl-tRNAr et N-formyltransferase (FMT; EC 2.1.2.9), is enough to prevent the incorporation into elongating peptide chains of the methionine esterified to tRNArICI. However, itis not yet known whether formylation is dispensable for Met-tRNAfMet initiator activity. In vitro studies with natural mRNA templates and Escherichia coli extracts showed that the formylation of Met-tRNA;4et enhanced translation rates (5, 12). In particular, the formyl group seemed to improve the efficiency of the selection of tRNAfMet by initiation factor IF2 (10, 24, 34). In vivo, it was found that the initiator activity of glutaminylated tRNAr et variants was related to their formylability (37). Moreover, the characterization of an fint strain pointed out the importance of tRNAP4et formylation in sustaining the rapid growth of E. coli (6).Besides determinants involved in its formylation, tRNAr et may carry other structural determinants involved in its functional specificity towards the initiation step. consisted of an in vivo assay in which variants of initiator or elongator tRNAs were exposed to various intracellular levels of FMT activity. The variants of tRNAfMe; and tRNA~et were constructed on the basis of the precise knowledge of the nucleotides governing the recognition of Met-tRNA1fet

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“…Thus (28) can initiate protein synthesis with glutamine, isoleucine, phenylalanine, valine, etc. (2,36). Thus, there is, a priori, no reason for restricting the initiating amino acid to methionine or the initiator codon to AUG.…”

Section: Critical Role Of Formylationmentioning

confidence: 99%

Initiator transfer RNAs

1994

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INTRODUCTIONHow does the cellular protein synthetic machinery distinguish initiator tRNA from all of the other tRNAs? What is the mechanism by which the initiator tRNA is sequestered for use exclusively in initiation of protein synthesis? Much interest has centered on questions of the sequence and/or structural information in the tRNA that is necessary for specifying the many distinctive properties of initiator tRNAs and the molecular basis by which the various proteins and/or components of the protein synthetic machinery utilize this information to distinguish initiator tRNAs from other tRNAs. Recent work, involving site-specific mutagenesis and analysis of the properties of the mutant tRNAs in vitro and in vivo, has led to identification of the sequence and structural features important in a eubacterial initiator tRNA. Here, I summarize this work and some of the implications of the results. Progress is also being made on initiator tRNAs from eukaryotic cytoplasm. This is included in another review (24).

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Initiation of in vivo protein synthesis with non-methionine amino acids

Cited by 67 publications

References 32 publications

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Initiation of Protein Synthesis in Mammalian Cells with Codons Other Than AUG and Amino Acids Other Than Methionine

Importance of formylability and anticodon stem sequence to give a tRNA(Met) an initiator identity in Escherichia coli

Initiator transfer RNAs

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