Abstract
2-Thioribothymidine (s(2)T) is a post-transcriptionally modified nucleoside of U54 specifically found in thermophilic bacterial tRNAs. The 2-thiocarbonyl group of s(2)T54 is known to be responsible for the thermostability of tRNA. The s(2)T54 content in tRNA varies depending on the cultivation temperature, a feature that confers thermal adaptation of protein synthesis in Thermus thermophilus. Little is known about the biosynthesis of s(2)T, including the sulfur donor, modification enzyme, and the tRNA structural requirements. To characterize 2-thiolation at position 54 in tRNA, we constructed an in vivo expression system using tRNA(Asp) with an altered sequence and a host-vector for T. thermophilus. We were able to detect in vivo activity of s(2)T54 thiolase using phenyl mercuric gel electrophoresis followed by Northern hybridization. 2-Thiolation at position 54 was identified in the precursor form of the tRNA, indicating that 2-thiolation precedes tRNA processing. To ascertain the elements that determine 2-thiolation in tRNA, systematic site-directed mutagenesis was carried out using the tRNA(Asp) gene. Conserved residues C56 and A58 were identified as major determinants of 2-thiolation, whereas tertiary interaction between the T and D loops and non-conserved nucleosides in the T loop were revealed not to be important for the reaction.
Highlights
A characteristic structural feature of tRNA is post-transcriptional modification
The tRNA melting temperature increased concomitantly with increases in the s2T content [6]. These findings indicate that 2-thiolation of T54 is responsible for the thermostability of T. thermophilus tRNA under diverse cultivation temperatures, thereby ensuring the thermal adaptation of protein synthesis
Sequence in E. coli and T. thermophilus Cells—To detect activity of the s2T [54]-thiolase in T. thermophilus cells and to investigate its recognition elements in tRNA in vivo, we first expressed an artificial tRNA gene encoded on a plasmid
Summary
Bacterial Strains and Media—T. thermophilus TTY1 (a ⌬leuB⌬pyrE strain derived from T. thermophilus HB27) [11] was used as the host strain throughout. A tRNA expression plasmid for T. thermophilus (pEx-Asp*) was constructed by excising the tRNAAsp* gene from pCR-XL-tRNAAsp* by double digestion with EcoRI and EcoRV and introducing it into the same sites of the E. coli-T. thermophilus shuttle vector pT8leuB [11]. Northern Hybridization—Total RNAs were extracted from cultured cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method as described in the literature [14] and subjected to electrophoretic analysis using a 10% polyacrylamide gel containing 7 M urea. The RNA was transferred onto a nylon membrane (Hybond-Nϩ, Amersham Biosciences) by blotting using TBE Buffer, which was hybridized with the 5Ј-32P-labeled DNA probes for tRNAAsp* or its precursor. To determine the initiation position of the transcription, the primer extension product was electrophoresed together with the dideoxy sequencing products of the template plasmid pCR-XL-tRNAAsp*(U8A) using the same primer (RT-1) on a 15% denaturing polyacrylamide sequencing gel
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