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PMID: 3277187 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 85 ·No. 4 ·1988-02-00 ·Pages 1033-7

Sampson JR, Uhlenbeck OC

Abstract

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.

MeSH Terms
Base Sequence DNA, Fungal/genetics DNA, Recombinant Molecular Sequence Data Nucleic Acid Conformation Nucleic Acid Denaturation Phenylalanine/metabolism Phenylalanine-tRNA Ligase/metabolism RNA, Fungal/genetics,metabolism RNA, Transfer, Amino Acid-Specific/genetics RNA, Transfer, Phe/genetics,metabolism Saccharomyces cerevisiae/genetics
Chemicals
DNA, Fungal DNA, Recombinant RNA, Fungal RNA, Transfer, Amino Acid-Specific RNA, Transfer, Phe Phenylalanine Phenylalanine-tRNA Ligase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Sampson J R
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Uhlenbeck O C
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.85.4.1033
Published
1988-02-00
Pages
1033-7
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC279695
Subset
IM
Grants
NIGMS NIH HHS · GM37552 · United States
External Links
PubMed source DOI: 10.1073/pnas.85.4.1033 PMC Full Text View journal details
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