Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

McClain, William H.; Foss, K.; Jenkins, Robert M.; Schneider, Jay

doi:10.1073/pnas.87.23.9260

Cited by 104 publications

(77 citation statements)

References 53 publications

(84 reference statements)

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“…A similar change in coding response was reported for tRNA Sec (19); anticodon changes led to efficient selenocysteine incorporation in response to all three termination codons (Table 4), even though the requirements for codon context are very different for Sec (UGA) and Pyl (UAG). Compared with the synthesis of wild-type LacZ protein from a lacZ ϩ construct, the absolute suppression efficiency of tRNA UCA Pyl is 20%, which is the same level as we observed (data not shown) and had been reported for the E. coli tRNA UCA Arg suppressor (20).…”

Section: Effect Of Mutations In Trna Pyl On In Vitro Charging By and supporting

confidence: 78%

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Ambrogelly¹,

Gundllapalli²,

Herring³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

119

126

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Pyrrolysine (Pyl), the 22nd naturally encoded amino acid, gets acylated to its distinctive UAG suppressor tRNA Pyl by the cognate pyrrolysyl-tRNA synthetase (PylRS). Here we determine the RNA elements required for recognition and aminoacylation of tRNA Pyl in vivo by using the Pyl analog N--cyclopentyloxycarbonyl-L-lysine. Forty-two Methanosarcina barkeri tRNA Pyl variants were tested in Escherichia coli for suppression of the lac amber A24 mutation; then relevant tRNA Pyl mutants were selected to determine in vivo binding to M. barkeri PylRS in a yeast three-hybrid system and to measure in vitro tRNA Pyl aminoacylation. tRNA Pyl identity elements include the discriminator base, the first base pair of the acceptor stem, the T-stem base pair G51:C63, and the anticodon flanking nucleotides U33 and A37. Transplantation of the tRNA Pyl identity elements into the mitochondrial bovine tRNA Ser scaffold yielded chimeric tRNAs active both in vitro and in vivo. Because the anticodon is not important for PylRS recognition, a tRNA Pyl variant could be constructed that efficiently suppressed the lac opal U4 mutation in E. coli. These data suggest that tRNA Pyl variants may decode numerous codons and that tRNA Pyl :PylRS is a fine orthogonal tRNA:synthetase pair that facilitated the late addition of Pyl to the genetic code.orthogonal tRNA ͉ suppression ͉ tRNA identity ͉ pyrrolysyl-tRNA synthetase ͉ aminoacyl-tRNA synthetase

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Section: Effect Of Mutations In Trna Pyl On In Vitro Charging By and supporting

confidence: 78%

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Ambrogelly¹,

Gundllapalli²,

Herring³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

119

126

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“…2). This nt is not directly involved in secondary or tertiary structure, but the substitution decreases the capacity of the tRNA to load arginine (27), thereby minimizing possible effects of the tRNA overproduction upon cell growth. In addition, the substitution permits the discrimination between the reporter and the genuine tRNAArg5 by hybridization (see Materials and Methods).…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

1994

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In gene expression studies, promoters are often fused to a protein-encoding reporter gene, the expression of which is then taken as an indirect measure of their strength. Here, we advocate the use of a tRNA reporter for the direct quantification of promoter strength. Using this method, we have studied the bacteriophage T7 gene 10 promoter in an E.coli strain that produces saturating amounts of T7 RNA polymerase. At 370C in aminoacid-glycerol medium, we show that this promoter ranks amongst the strongest known, directing ca 1.1 transcription events per second, 2.2-fold more than the promoters for rRNA operons, or 15-fold more than the induced lac promoter. Surprisingly, compared to the lac promoter, the T7 promoter is far less efficient in driving the expression of protein-encoding genes such as cat, neo or lacZ. Therefore, the polypeptide yield per transcript is lower when the T7 RNA polymerase is used instead of the E.coli RNA polymerase. The former enzyme travels faster than the translating ribosomes, and we suggest that this desynchronization lowers the polypeptide yield per transcript. INTRODUCTIONBacteriophages T3, T7 and SP6 encode closely related RNA polymerases which transcribe the viral genome from highly specific promoters, during the late phase of infection (1). The prototype of the group, the T7 RNA polymerase, can be stably expressed in E. coli, and genes that are fused to a T7 late promoter can be expressed to high levels when introduced in such hosts (2, 3). In spite of this practical bearing, relatively little is known about the in vivo behaviour of these RNA polymerases. Indirect evidence suggests that the T7 RNA polymerase interacts very efficiently with its promoter in E. coli (4), whereas in vitro the interaction appears relatively weak (1,5). The enzyme has a very high turnover for the polymerisation of ribonucleotides, both in vitro (6) and in vivo (7). As a result, in E. coli it runs far ahead of the ribosomes which translate its nascent transcript, a situation

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“…In contrast, the most conspicuous feature of the substrate recognition of colicin D is that only tRNAs Arg and all isoaccepting tRNAs Arg are susceptible, just as in the case of the cognate tRNA recognition by arginyl-tRNA synthetase (ArgRS). The main identity elements needed for the recognition of tRNAs Arg by ArgRS were eluci- dated to be A20, C35, and the discriminator (29)(30)(31). Detailed analyses are now in progress to determine whether colicin D uses a similar recognition mechanism as ArgRS.…”

Section: Discussionmentioning

confidence: 99%

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

Tomita

Ogawa

Uozumi

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

158

143

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Colicin D has long been thought to stop protein synthesis in infected Escherichia coli cells by inactivating ribosomes, just like colicin E3. Here, we show that colicin D specifically cleaves tRNAs Arg including four isoaccepting molecules both in vivo and in vitro. The cleavage occurs in vitro between positions 38 and 39 in an anticodon loop with a 2 ,3 -cyclic phosphate end, and is inhibited by a specific immunity protein. Consistent with the cleavage of tRNAs Arg , the RNA fraction of colicin-treated cells significantly reduced the amino acid-accepting activity only for arginine. Furthermore, we generated a single mutation of histidine in the C-terminal possible catalytic domain, which caused the loss of the killing activity in vivo together with the tRNA Arg -cleaving activity both in vivo and in vitro. These findings show that colicin D directly cleaves cytoplasmic tRNAs Arg , which leads to impairment of protein synthesis and cell death. Recently, we found that colicin E5 stops protein synthesis by cleaving the anticodons of specific tRNAs for Tyr, His, Asn, and Asp. Despite these apparently similar actions on tRNAs and cells, colicins D and E5 not only exhibit no sequence homology but also have different molecular mechanisms as to both substrate recognition and catalytic reaction.C olicins are plasmid-encoded proteins that are toxic to Escherichia coli cells that do not have the same plasmid or a cognate Col plasmid (1-5). Most colicins are produced in response to SOS-inducing signals and are secreted into the medium. After binding to cell-surface receptors on sensitive cells, they are translocated across the membrane and then exert their final cytotoxic activities, which are attributable to their C-terminal domains. Two major modes of toxicity are well known; colicins A, B, E1, Ia, Ib, K, and N are ion-channel formers attacking the cytoplasmic membrane, and colicins E2 to E9 are nucleases. In the latter group, E2, E7, E8, and E9 are DNases, and E3 is a special kind of RNase that cleaves 16S rRNA within ribosomes.Colicins E4 to E6 quickly stop amino acid incorporation in treated cells (6), suggesting impairment of protein synthesis analogous in the established case of E3, which specifically cleaves 16S-RNA at the 49th bond from the 3Ј end leading to inactivation of ribosomes (7-9). This is also the case with colicin D, which Timmis and Hedges have characterized as to the physiological response of treated cells (10, 11), although the actual molecular basis of the cytotoxic effect of colicin D, as well as those of E4 to E6, remained to be elucidated. Colicins E4 and E6 proved to be E3-homologs and showed comparable activity toward ribosomes (ref. 12; GenBank accession number X63621; Y. Gunji, M. Ohno, T.O., H.M., and T.U., unpublished data), but colicin E5 exhibits no similarity to E3 in the C-terminal active domain. We recently showed that colicin E5 comprises a third category of nuclease-type colicins, which does not attack ribosomes but specific tRNAs (13). E5 is a novel RNase that cleaves the anticodons...

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Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

Cited by 104 publications

References 53 publications

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

Pyrrolysine is not hardwired for cotranslational insertion at UAG codons

The use of a tRNA as a transcriptional reporter: the T7 late promoter is extremely efficient inEscherichia colibut its transcripts are poorly expressed

A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops

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