Transfer RNA recognition by class I lysyl-tRNA synthetase from the Lyme disease pathogen Borrelia burgdorferi

Borrelia burgdorferi and other spirochetes contain a class I lysyl-tRNA synthetase (LysRS), in contrast to most eubacteria that have a canonical class II LysRS. We analyzed tRNA(Lys) recognition by B. burgdorferi LysRS, using two complementary approaches. First, the nucleotides of B. burgdorferi tRNA(Lys) in contact with B. burgdorferi LysRS were determined by enzymatic footprinting experiments. Second, the kinetic parameters for a series of variants of the B. burgdorferi tRNA(Lys) were then determined during aminoacylation by B. burgdorferi LysRS. The identity elements were found to be mostly located in the anticodon and in the acceptor stem. Transplantation of the identified identity elements into the Escherichia coli tRNA(Asp) scaffold endowed lysylation activity on the resulting chimera, indicating that a functional B. burgdorferi lysine tRNA identity set had been determined.     

Human Tyrosine tRNA Is Also Internally Cleavable by E. coli Ribonuclease P RNA Ribozyme in Vitro

Transfer RNA is an essential molecule for biological system, and each tRNA molecule commonly has a cloverleaf structure. Previously, we experimentally showed that some Drosophila tRNA (tRNA^Ala, tRNA^His, and tRNA_i ^Met) molecules fit to form another, non-cloverleaf, structure in which the 3'-half of the tRNA molecules forms an alternative hairpin, and that the tRNA molecules are internally cleaved by the catalytic RNA of bacterial ribonuclease P (RNase P). Until now, the hyperprocessing reaction of tRNA has only been reported with Drosophila tRNAs. This time, we applied the hyperprocessing reaction to one of human tRNAs, human tyrosine tRNA, and we showed that this tRNA was also hyperprocessed by E. coli RNase P RNA. This tRNA is the first example for hyperprocessed non-Drosophila tRNAs. The results suggest that the hyperprocessing reaction can be a useful tool to detect destablized tRNA molecules from any species.     

Evidence for Late Resolution of the AUX Codon Box in Evolution

Recognition strategies for tRNA aminoacylation are ancient and highly conserved, having been selected very early in the evolution of the genetic code. In most cases, the trinucleotide anticodons of tRNA are important identity determinants for aminoacylation by cognate aminoacyl-tRNA synthetases. However, a degree of ambiguity exists in the recognition of certain tRNA^Ile isoacceptors that are initially transcribed with the methionine-specifying CAU anticodon. In most organisms, the C34 wobble position in these tRNA^Ile precursors is rapidly modified to lysidine to prevent recognition by methionyl-tRNA synthetase (MRS) and production of a chimeric Met-tRNA^Ile that would compromise translational fidelity. In certain bacteria, however, lysidine modification is not required for MRS rejection, indicating that this recognition strategy is not universally conserved and may be relatively recent. To explore the actual distribution of lysidine-dependent tRNA^Ile rejection by MRS, we have investigated the ability of bacterial MRSs from different clades to differentiate cognate tRNA_CAU^Met from near-cognate tRNA_CAU^Ile. Discrimination abilities vary greatly and appear unrelated to phylogenetic or structural features of the enzymes or sequence determinants of the tRNA. Our data indicate that tRNA^Ile identity elements were established late and independently in different bacterial groups. We propose that the observed variation in MRS discrimination ability reflects differences in the evolution of genetic code machineries of emerging bacterial clades.     

膜上tRNA结合蛋白的分离与初步鉴定

用ＴｒｉｔｏｎＸ－１１４分相法分离啤酒酵母的膜总蛋白,经过酵母ｔＲＮＡ分子交联的Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析,用０－０．８ｍｏｌ／Ｌ（ＮＨ４０２ＳＯ４梯度缓冲液洗脱ｔＲＮＡ结合的蛋白质。凝胶阻滞电泳实验室鉴定出两种主要的与ｔＲＮＡ分子特异性结合的蛋白质。     

Transfer RNA genes in pieces

The short genes encoding transfer RNA (tRNA) molecules are highly conserved in both sequence and structure, reflecting the central role of tRNA in protein biosynthesis. The frequent occurrence of fragmented intron-containing tRNAs that require processing to form contiguous molecules is therefore surprising. Recent discoveries of permuted and split tRNA genes have added to the apparent creativity of nature regarding the organization of these fragmented genes. Here, we provide an overview of the various types of fragmented tRNA genes and examine the hypothesis that the integration of mobile genetic elements--including viruses and plasmids--established such genes in pieces.     

Conservation of plant cytoplasmic tRNA structure. Nucleotide sequence of rape tRNAPhe

The primary structure of rape seeds tRNAPhe has been determined. It is identical to that of wheat germ, pea and barley tRNAsPhe, and of the minor tRNAPhe species of yellow lupin seeds.     

Anticodon conformation and accessibility in wild-type and suppressor tryptophan tRNA from E. coli.

The association between Trp-tRNA and Pro-tRNA, which have complementary anticodon sequences, has been used as a probe of anticodon conformation. It is unaffected, however, by the base change in the D-stem present in UGA-suppressor Trp-tRNA. This does not support the hypothesis that UGA suppression depends upon a conformational change induced in the anticodon. The stable denatured form of wild-type Trp-tRNA no longer interacts with Pro-tRNA; the structure of the anticodon region must therefore be quite different in the denatured form.     

Effects of dilute HCl on yeast tRNAPhe and E. coli tRNAfMet1

HCl treatment of yeast tRNA^Phe under conditions generally used for excision of `Y' base results in structure and conformation changes as monitored by line widths in the PMR spectra at 220 MHz and by optical rotation. Like exposure of E. coli tRNA^fMet₁ causes similar changes in the PMR spectra and optical rotation although no residues are eliminated. Electrophoresis in polyacrylamide gels provides evidence for aggregation in HCl-treated tRNA^fMet₁. One must thus consider a general effect of HCl exposure as well as possible residue removal in assessing induced structural and conformation changes in tRNA.     

The Chemical Synthesis of E. coli tRNALys Anticodon Loop Fragment and Its Analogues

Abstract

E. coli tRNA^Lys anticodon loop fragment (Umnm⁵sUUUt⁶A) 1 and its analogues 2–6 were synthesized by the classical phosphotriester approach in solution. The preparation of suitably protected derivatives of N⁶-threonylcarbamoyladenosine 18 is also described.     

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.     

The Nature of the Nucleotide at the 5' Side of the tRNA Su9 Anticodon Affects UGA Suppression in Escherichia coli

In Escherichia coli a UGA codon can be efficiently suppressedby a suppressor tRNA^Trp called Su9. Here, we show that the levelof UGA suppression is determined by the nature of the nucleotideat the 5' side of the anticodon of the suppressor (position33). UGA suppression occurs when a pyrimidine residue is locatedin position 33 of the tRNA, and suppression is more efficientwith a U than with a C in this position. On the other hand,when a purine residue is located at this position UGA suppressionis extremely low. These results show that in the case of tRNASu9, the UGA codon context effect does not require base pairingbetween the nucleotide at the 3' side of the codon and the 5'side of the anticodon.     

Axoplasmic Transport of Transfer RNA in the Chick Optic System

It has previously been shown that 4S RNA is transported in the optic nerve of the chick, but that no movement of rRNA can be detected. The 4S component behaved as though it were composed mainly of transfer RNA (tRNA), but the possibility remained that it could contain significant amounts of material resulting from RNA degradation. The transport of this 4S component has been examined in more detail to determine its nature. In addition, the transported material was examined to establish whether the transport of tRNA is a general phenomenon or that there are only a limited number of species involved. This was done using the same principles applied in the previous study; i.e., the specific activities of separated 4S RNA species appearing in the optic tectum 4 days after intraocular injection of [3H]uridine were compared with that of 5S RNA, a nontransported species. The separation was accomplished using 2.8-5-10-17% slab polyacrylamide gels, and 18 separate regions of 4S species could be identified. The results show that at least most, if not all 4S RNA species are transported. In a separate series of experiments the 4S RNA was aminoacylated and again separated on slab gels. In this instance, the RNA was labelled with [3H]uridine and the aminoacyl component with [14C]amino acids. Gel profiles of these dual-labelled components showed excellent correspondence between the two labels, demonstrating that 4S RNA species could be aminoacylated and were therefore tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)     

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.