Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem

Chan Ping Lee, Baik L. Seong, Uttam L. RajBhandary

Research output: Contribution to journalArticle

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Abstract

We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A "mismatch" in E. coli initiator tRNA. Mutation of this C.A to U:A, a weak base pair, or U.G, a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kapp m by 495-fold. Mutation of the second base-pair G2:C71 to U2:A71 lowers Vmax/Kapp m by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kapp m by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and TψC stem have little or no effect on formylation.

Original languageEnglish
Pages (from-to)18012-18017
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number27
Publication statusPublished - 1991 Sep 25

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methionyl-tRNA formyltransferase
RNA, Transfer, Met
Base Pairing
Escherichia coli
Anticodon
Transfer RNA
Amino Acids
Mutagenesis
Mutation
Discriminators
Kinetic parameters
Genes
formylmethionine-tRNA

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

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title = "Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem",
abstract = "We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A {"}mismatch{"} in E. coli initiator tRNA. Mutation of this C.A to U:A, a weak base pair, or U.G, a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kapp m by 495-fold. Mutation of the second base-pair G2:C71 to U2:A71 lowers Vmax/Kapp m by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kapp m by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and TψC stem have little or no effect on formylation.",
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Structural and sequence elements important for recognition of Escherichia coli formylmethionine tRNA by methionyl-tRNA transformylase are clustered in the acceptor stem. / Lee, Chan Ping; Seong, Baik L.; RajBhandary, Uttam L.

In: Journal of Biological Chemistry, Vol. 266, No. 27, 25.09.1991, p. 18012-18017.

Research output: Contribution to journalArticle

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N2 - We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A "mismatch" in E. coli initiator tRNA. Mutation of this C.A to U:A, a weak base pair, or U.G, a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kapp m by 495-fold. Mutation of the second base-pair G2:C71 to U2:A71 lowers Vmax/Kapp m by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kapp m by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and TψC stem have little or no effect on formylation.

AB - We show that the structure and/or sequence of the first three base pairs at the end of the amino acid acceptor stem of Escherichia coli initiator tRNA and the discriminator base 73 are important for its formylation by E. coli methionyl-tRNA transformylase. This conclusion is based on mutagenesis of the E. coli initiator tRNA gene followed by measurement of kinetic parameters for formylation of the mutant tRNAs in vitro and function in protein synthesis in vivo. The first base pair found at the end of the amino acid acceptor stem in all other tRNAs is replaced by a C.A "mismatch" in E. coli initiator tRNA. Mutation of this C.A to U:A, a weak base pair, or U.G, a mismatch, has little effect on formylation, whereas mutation to C:G, a strong base pair, has a dramatic effect lowering Vmax/Kapp m by 495-fold. Mutation of the second base-pair G2:C71 to U2:A71 lowers Vmax/Kapp m by 236-fold. Replacement of the third base-pair C3:G70 by U3:A70, A3:U70, or G3:C70 lowers Vmax/Kapp m by about 67-, 27-, and 30-fold, respectively. Changes in the rest of the acceptor stem, dihydrouridine stem, anticodon stem, anticodon sequence, and TψC stem have little or no effect on formylation.

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