Abstract
Mutations in selC, which reduce the 8-base pair aminoacyl-acceptor helix to the canonical 7-base pair length (tRNA(Sec)(delAc] or which replace the extra arm of tRNA(Sec) by that of a serine acceptor tRNA species (tRNA(Sec)(ExS), block the function in selenoprotein synthesis in vivo (Baron, C., Heider, J., and Böck, A. (1990) Nucleic Acids Res. 18, 6761-6766). tRNA(Sec), tRNA(Sec)(delAc), and tRNA(Sec)(ExS) were purified and analyzed for their interaction with purified seryl-tRNA synthetase, selenocysteine synthase and translation factors SELB and EF-Tu. It was found that seryl-tRNA synthetase displays 10-fold impaired Km and Kcat values for tRNA(Sec) in comparison to tRNA(Ser), decreasing the overall charging efficiency (Kcat/Km) of tRNA(Sec) to 1% of that characteristic for tRNA(Ser). tRNA(Sec)(ExS) was a less efficient substrate for the enzyme (Kcat/Km 0.2% of the tRNA(Ser) value) whereas the tRNA(Ser)(delAc) variant was charged with an approximately 2-3-fold improved rate compared to wild-type tRNA(Sec). Both mutant tRNA variants, when charged with L-serine, were able to interact with selenocysteine synthase to give rise to selenocysteyl-tRNA with tRNA(Sec)(ExS) being as efficient as wild-type tRNA(Sec). Seryl-tRNA(Sec)(delAc), on the other hand, was selenylated very slowly. Reduction of the length of the aminoacyl-acceptor stem to 7 base pairs prevented the interaction with translation factor SELB but allowed binding to EF-Tu, irrespective of whether tRNA(Sec)(delAc) was charged with serine or selenocysteine. The aminoacyl-acceptor helix of tRNA(Sec), therefore, is a major determinant directing binding to SELB and precluding interaction with EF-Tu.
Highlights
The Length of the Aminoacyl-acceptor Stem of the Selenocysteinespecific tRNASecof Escherichia coli Is the Determinant for Binding to Elongation Factors SELB or Tu*
We present a detailed analysis of the i n vitro interactions of these mutant tRNAswith seryl-tRNA synthetase, selenocysteine synthase, SELB protein, and Elongation Factor Tu (EF-Tu)
Materials tRNA'"", the selenocysteine-specific tRNA species, is the keymolecule in selenoprotein synthesis in Escherichia coli and other microorganisms.This tRNA undergoes a number of macromolecular interactions during the process of selenocysteine biosynthesis and insertion into the growing polypeptide chain
Summary
Materials tRNA'"", the selenocysteine-specific tRNA species, is the keymolecule in selenoprotein synthesis in Escherichia coli and other microorganisms (for review, seeRef. 1).This tRNA undergoes a number of macromolecular interactions during the process of selenocysteine biosynthesis and insertion into the growing polypeptide chain. Potassium borohydride was purchased from Sigma and ATP, GTP, pyruvate kinase, and pyrophosphatase were obtained from Boehringer Mannheim. Overproduction and Purification otf R N A tRNAs were overproduced in cells of E. coli strain FM460 (AselC) carrying plasmids pSU2719 (tRNASer1()9) or pUC19 [10] derivatives harboring the wild-type selC gene or mutant derivatives delAc or ExS thereof [8].The tRNA genes are under the control of the lac promoter. EF-Tu was purified from E. coli FM460 cells following the procedure of Wenzig and Schleifer [14]
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