Ligation of endogenous tRNA 3' half molecules to their corresponding 5' halves via 2'-phosphomonoester,3',5'-phosphodiester bonds in extracts of Chlamydomonas

tRNA preparations from Chlamydomonas and wheat germ contain small amounts of tRNA 5' halves and corresponding 3' halves. Incubation of cell-free extracts from the two sources with [γ-³²P]ATP yielded 5'-³²P-labeled tRNA 3' halves which were joined to their corresponding 5' counterparts to form mature tRNA containing 2'-phosphomonoester,3', 5'-phosphodiester bonds. tRNA 3' halves labelled with T4 kinase were purified, sequenced and also joined to their 5' counterparts. It is proposed that these tRNA halves may be intermediates of the tRNA splicing process, and that the RNA kinase and ligase activities observed here are part of the tRNA splicing complex.     

Altered tRNA characteristics and 3' maturation in bacterial symbionts with reduced genomes

Translational efficiency is controlled by tRNAs and other genome-encoded mechanisms. In organelles, translational processes are dramatically altered because of genome shrinkage and horizontal acquisition of gene products. The influence of genome reduction on translation in endosymbionts is largely unknown. Here, we investigate whether divergent lineages of Buchnera aphidicola, the reduced-genome bacterial endosymbiont of aphids, possess altered translational features compared with their free-living relative, Escherichia coli. Our RNAseq data support the hypothesis that translation is less optimal in Buchnera than in E. coli. We observed a specific, convergent, pattern of tRNA loss in Buchnera and other endosymbionts that have undergone genome shrinkage. Furthermore, many modified nucleoside pathways that are important for E. coli translation are lost in Buchnera. Additionally, Buchnera’s A + T compositional bias has resulted in reduced tRNA thermostability, and may have altered aminoacyl-tRNA synthetase recognition sites. Buchnera tRNA genes are shorter than those of E. coli, as the majority no longer has a genome-encoded 3'' CCA; however, all the expressed, shortened tRNAs undergo 3′ CCA maturation. Moreover, expression of tRNA isoacceptors was not correlated with the usage of corresponding codons. Overall, our data suggest that endosymbiont genome evolution alters tRNA characteristics that are known to influence translational efficiency in their free-living relative.     

Evolution of tRNAs and tRNA genes in Acholeplasma laidlawii.

The genes for 22 tRNA species from Acholeplasma laidawii, belonging to the class Mollicutes (Mycoplasmas), have been cloned and sequenced. Sixteen genes are organized in 3 clusters consisting of eleven, three and two tRNA genes, respectively, and the other 6 genes exist as a single gene. The arrangement of tRNA genes in the 11-gene, the 3-gene and the 2-gene clusters reveals extensive similarity to several parts of the 21-tRNA or 16-tRNA gene cluster in Bacillus subtilis. The 11-gene cluster is also similar to the tRNA gene clusters found in other mycoplasma species, the 9-tRNA gene cluster in M.capricolum and in M.mycoides, and the 10-tRNA gene cluster in Spiroplasma meliferm. The results suggest that the tRNA genes in mycoplasmas have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to mycoplasmas and B.subtilis. The anticodon sequences including base modifications of 15 tRNA species from A.laidlawii were determined. The anticodon composition and codon-recognition patterns of A.laidlawii resemble those of Bacillus subtilis rather than those of other mycoplasma species.     

Conserved signals in the 5' flanking region of eukaryotic nuclear tRNA genes.

The statistical analysis of 5' flanking regions of eukaryotic tRNA genes was done. The analysis of nucleotides in the sequence of fungi and invertebrates showed a high content of A and T in the flanking regions versus coding regions where G and C dominate. In contrast to these results in vertebrates sequences the preferences of any nucleotide in flanking regions was not observed. The analysis of tetrads showed five conserved signals: TTGT, (T/A)(T/A)ATA, A(C/T)(C/A)A in the tRNA genes of fungi, (A/T)TGA of invertebrates and (A/T)GAG of vertebrates. The analysis of 3' flanking regions did not show any conserved signals except well known poly-T tracks.     

Coexistence of bacterial leucyl-tRNA synthetases with archaeal tRNA binding domains that distinguish tRNALeu in the archaeal mode

Leucyl-tRNA (transfer RNA) synthetase (LeuRS) is a multi-domain enzyme, which is divided into bacterial and archaeal/eukaryotic types. In general, one specific LeuRS, the domains of which are of the same type, exists in a single cell compartment. However, some species, such as the haloalkaliphile Natrialba magadii, encode two cytoplasmic LeuRSs, NmLeuRS1 and NmLeuRS2, which are the first examples of naturally occurring chimeric enzymes with different domains of bacterial and archaeal types. Furthermore, N. magadii encodes typical archaeal tRNA^Leus. The tRNA recognition mode, aminoacylation and translational quality control activities of these two LeuRSs are interesting questions to be addressed. Herein, active NmLeuRS1 and NmLeuRS2 were successfully purified after gene expression in Escherichia coli. Under the optimized aminoacylation conditions, we discovered that they distinguished cognate NmtRNA^Leu in the archaeal mode, whereas the N-terminal region was of the bacterial type. However, NmLeuRS1 exhibited much higher aminoacylation and editing activity than NmLeuRS2, suggesting that NmLeuRS1 is more likely to generate Leu-tRNA^Leu for protein biosynthesis. Moreover, using NmLeuRS1 as a model, we demonstrated misactivation of several non-cognate amino acids, and accuracy of protein synthesis was maintained mainly via post-transfer editing. This comprehensive study of the NmLeuRS/tRNA^Leu system provides a detailed understanding of the coevolution of aminoacyl-tRNA synthetases and tRNA.     

The use of cloned tRNA genes for the purification and measurement of specific tRNAs

We have previously reported the ability of a cloned tRNAMeti gene (pt145) to bind tRNAMeti specifically [5]. In this paper, we show that a pBR322 plasmid containing the tRNAAsn gene of Xenopus (pt38 - donated by Stuart Clarkson) will specifically bind to mouse tRNAAsn when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNAAsn by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNAAsn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAAsn/cell. The fraction of newly synthesized tRNA representing tRNAAsn or tRNAMeti was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.     

Mutant enzymes and tRNAs as probes of the glutaminyl-tRNA synthetase: tRNA interaction

This paper focuses on several aspects of the specificity of mutants of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) and tRNA^Gln. Temperature-sensitive mutants located in glnS, the gene for GlnRS, have been described previously. The mutations responsible for the temperature-sensitive phenotype were analyzed, and pseudorevertants of these mutants isolated and characterized. The nature of these mutations is discussed in terms of their location in the three-dimensional structure of the tRNA^Gln: GlnRS complex. In order to characterize the specificity of the aminoacylation reaction, mutant tRNA^Gln species were synthesized with either a 2′-deoxy AMP or 3′-deoxy AMP as their 3′-terminal nucleotide. Subsequent assays for aminoacylation and ATP/PP_i exchange activity established the esterification of glutamine to the 2′-hydroxyl of the terminal adenosine: there is no glutaminylation of the 3′-OH group. This correlates with the classification of GlnRS as a class I aminoacyl-tRNA synthetase. Mutations in tRNA^Gln are discussed which affect the recognition of GlnRS and the current concept of glutamine identity in E coli is reviewed.     

Physical and functional interaction between archaeal single-stranded DNA-binding protein and the 5'-3' nuclease NurA

NurA is a novel 5′-3′ exonuclease that is closely linked to Mre11 and Rad50 homologues in most thermophilic archaea. We report a physical and functional interaction between NurA (StoNurA) and single-stranded DNA-binding protein (StoSSB) from the hyperthermophilic archaeon Sulfolobus tokodaii. StoSSB was identified as a novel StoNurA-interacting protein by pull-down assay using Ni-NTA agarose beads and MALDI-TOF mass spectrometry. The direct interaction between StoNurA and StoSSB was further confirmed by yeast two-hybrid and co-immunoprecipitation analysis. The interaction was supposed to have functional significance because it was found that StoSSB inhibited the 5′-3′ ssDNA and dsDNA exonuclease and ssDNA endonuclease activities of StoNurA. Our results suggest that NurA may function closely together with SSB in DNA transactions in archaea.     

Transfer RNA genes in Drosophila mitochondrial DNA: related 5' flanking sequences and comparisons to mammalian mitochondrial tRNA genes.

Genes for tRNAgly and tRNAserUCN have been identified within sequences of mtDNA of Drosophila yakuba. The tRNAgly gene lies between the genes for cytochrome c oxidase subunit III and URF3, and all three of these genes are contained in the same strand of the mtDNA molecule. The tRNAserUCN gene is adjacent to the URF1 gene. These genes are contained in opposite strands of the mtDNA molecule and their 3' ends overlap. The structures of the tRNAgly and tRNAserUCN genes, and of the four tRNA genes of D. yakuba mtDNA reported earlier (tRNAile, tRNAgln, tRNAf-met and tRNAval) are compared to each other, to non-organelle tRNAs, and to corresponding mammalian mitochondrial tRNA genes. Within 19 nucleotides upstream from the 5' terminal nucleotide of each of the Drosophila mitochondrial tRNAgly, tRNAserUCN, tRNAile, tRNAgln and tRNAf-met genes occurs the sequence 5'TTTATTAT, or a sequence differing from it by one nucleotide substitution. Upstream from this octanucleotide sequence, and separated from it by 3, 4 and 11 nucleotides, respectively, in the 5' flanking regions of the tRNAile, tRNAserUCN and tRNAgly genes occurs the sequence 5'GATGAG.     

Sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3' NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.