We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 microg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).
We have determined that lOSa RNA (one of the small stable RNAs found in Escherichia cobl) has an interesting structural feature: the 5' end and the 3' end of lOSa RNA can be arranged in a s ture that is equivalent to a half-molecule (acceptor stem and TFC stem-loop) of alanine tRNA of E. coli. Primer-extension analysis of lOSa RNA extracted from a bacterial mutant with temperature-sensitive RNase P function revealed that the precursor to lOSa RNA (pre-lOSa RNA) is folded into a pre-tRNA-like structure in vivo such that it can be cleaved by RNase P to generate the 5' end of the mature 10Sa RNA. The purified 10Sa RNA can be charged with alanine in vitro. Disruption of the gene encoding lOSa RNA (ssrA) caused a reduction in the rate of cell growth, which was especaly apparent at 45C, and a reduction in motility on se d agar. These phenotypic characteristics of the deletion strain (AssrA) allowed us to investigate the effects of some mutations in lOSa RNA in vivo, although the exact function of lOSa RNA still remains unclear. When the G-U pair (G3-U357) in lOSa RNA, which may be equivalent to the determinant G-U pair of alanine tRNA, was changed to a GA or G-C pair, the ability to complement the phenotypic mutations of the AssrA strain was lost. Furthermore, this inability to complement the mutant phenotypes that was caused by the substitution of the determinant bases by a GA pair could be overcome by the introduction of a gene encoding alanyl-tRNA synthetase (aaS) on a multicopy plasmid. The evidence suggests that the proposed structural features of lOSa RNA are indeed manifested in vivo.lOSa RNA is one of the small stable RNAs that is found in Escherichia coli. It was first identified as lOS RNA, which was actually a mixture of lOSa RNA and Ml RNA (originally designated lOSb RNA) (1, 2). Subsequently, Ml RNA was shown to be a "catalytic RNA" (3), and it has been extensively studied from both a structural and a functional perspective (4). However, in contrast to Ml RNA, lOSa RNA has not been the focus of much recent attention, and very little is known about either its structure or its function.In our previous study, we found that the ssrA gene that encodes lOSa RNA of E. coli is included in the genomes of phages 438, 439, and 440 of Kohara's library (5), which corresponds to 56.5 minutes on the linkage map of the chromosome (6). We cloned a 2.2-kb fragment that covers the overlapping region of these phages into a plasmid vector and determined the nucleotide sequence of the fragment. § We also purified lOSa RNA from E. coli strain W3110 and determined its nucleotide sequence from both the 5' and the 3' end.The nucleotide sequence of the ssrA gene from E. coli has already been reported by Chauhan and Apirion (7). In comparing our sequences with the published sequence, we noticed an interesting structural feature of lOSa RNA, as presented in Fig. 2-namely, 7 nucleotides at the 5' end and 28 nucleotides at the 3' end can be arranged in a structure that is equivalent to a half-molecule of tRNA. This half-mole...
An unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography-mass spectrometry (LC-MS) analysis of the products. This coupled transcription-translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.
N7-methylguanine at position 46 (m7G46) in tRNA is produced by tRNA (m7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant (ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m7G46 modification in tRNAPhe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m1G37, suggesting that the m7G46 positively affects their formations. Although the lack of the m7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNAPhe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNAPhe and tRNAIle. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m7G46 modification supports introduction of other modifications.
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