Transfer RNA Identity Change in Anticodon Variants of E. coli tRNAPhe in Vivo

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA^Phe. Constructing different anticodon mutants of E. coli tRNA^Phe by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA^Phe; the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA^Phe genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA^Phe are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.     

Characterisation of fission yeast alp11 mutants defines three functional domains within tubulin-folding cofactor B

The proper folding of tubulins prior to their incorporation into microtubules requires a group of conserved proteins called cofactors A to E. In fission yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability. Here we show that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11^B protein (Alp11_1–118). We isolated a tRNA^Trp gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon. The truncated Alp11_1–118 protein lacks the C-terminal half of Alp11^B, consisting of a central coiled-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions. Both of these domains are required for the maintenance of microtubule architecture in vivo. Detailed functional analyses lead us to propose that Alp11^B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular α-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to α-tubulin. Received: 29 November 1999 / Accepted: 18 April 2000     

Knocking out a specific tRNA species within unfractionated Escherichia coli tRNA by using antisense (complementary) oligodeoxyribonucleotides

Methods for the preparation of an Escherichia coli tRNA mixture lacking one or a few specific tRNA species can be the basis for future applications of cell-free protein synthesis. We demonstrate here that virtually a single tRNA species in a crude E. coli tRNA mixture can be knocked out by an antisense (complementary) oligodeoxyribonucleotide. One out of five oligomers complementary to tRNA^Asp blocked the aspartylation almost completely, while minimally affecting the aminoacylation with other 13 amino acids tested. This `knockout' tRNA behaved similarly to the untreated tRNA in a cell-free translation of an mRNA lacking Asp codons.     

A Reexamination of the Phylogenetic Position of Callimico (Primates) Incorporating New Mitochondrial DNA Sequence Data

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNA^His, tRNA^Ser, and tRNA^Leu genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets (Callithrix and Cebuella) and the tamarins (Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico. Received: 20 March 1997 / Accepted: 17 December 1997     

A cognate tRNA specific conformational change in glutaminyl-tRNA synthetase and its implication for specificity.

Conformational changes that occur upon substrate binding are known to play crucial roles in the recognition and specific aminoacylation of cognate tRNA by glutaminyl-tRNA synthetase. In a previous study we had shown that glutaminyl-tRNA synthetase labeled selectively in a nonessential sulfhydryl residue by an environment sensitive probe, acrylodan, monitors many of the conformational changes that occur upon substrate binding. In this article we have shown that the conformational change that occurs upon tRNA(Gln) binding to glnRS/ATP complex is absent in a noncognate tRNA tRNA(Glu)-glnRS/ATP complex. CD spectroscopy indicates that this cognate tRNA(Gln)-induced conformational change may involve only a small change in secondary structure. The Van't Hoff plot of cognate and noncognate tRNA binding in the presence of ATP is similar, suggesting similar modes of interaction. It was concluded that the cognate tRNA induces a local conformational change in the synthetase that may be one of the critical elements that causes enhanced aminoacylation of the cognate tRNA over the noncognate ones.     

Nonsense and missense translational suppression in plant cells mediated by tRNALys

A nuclear tRNA^Lys gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (CAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gln, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA^Lys and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA _CUA ^Lys from non-replicative vectors promoted 10–40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA^Lys suppressor genes from a geminivirus vector capable of replication promoted 30–80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gln and Glu codons.     

多靶向RNA干扰技术在基因治疗中的应用与前景

RNA干扰(RNA interference,RNAi)通过转录后基因沉默效应特异性抑制靶基因的表达,其沉默机制的高效性、特异性及稳定性使这项技术成为生物医学领域研究基因治疗的重要工具。阐述RNAi技术的特点和RNAi疗法的现状,特别是多靶小干扰RNA(small interference RNA,siRNA)目前的发展态势及其各种结构性修饰,通过使用这些结构修饰的siRNA提高基因沉默的效率,将有助于提高疗效。但该技术在广泛应用于临床之前,仍存在一些亟待解决的问题与面临的挑战,需进一步研究。     

mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNA^Met (iMet) through 5′–3′ exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR-regulated iMet degradation in mammalian cells.     

Optimization protein productivity of human interleukin-2 through codon usage,gene copy number and intracellular tRNA concentration in CHO cells

Transfer RNA (tRNA) abundance is one of the critical factors for the enhancement of protein productivity in prokaryotic and eukaryotic hosts. Gene copy number of tRNA and tRNA codon usage bias are generally used to match tRNA abundance of protein-expressing hosts and to optimize the codons of recombinant proteins. Because sufficient concentration of intracellular tRNA and optimized codons of recombinant proteins enhanced translation efficiency, we hypothesized that sufficient supplement of host’s tRNA improved protein productivity in mammalian cells. First, the small tRNA sequencing results of CHO-K1 cells showed moderate positive correlation with gene copy number and codon usage bias. Modification of human interleukin-2 (IL-2) through codons with high gene copy number and high codon usage bias (IL-2 HH, modified on Leu, Thr, Glu) significantly increased protein productivity in CHO-K1 cells. In contrast, modification through codons with relatively high gene copy number and low codon usage bias (IL-2 HL, modified on Ala, Thr, Val), or relatively low gene copy number and low codon usage bias (IL-2 LH, modified on Ala, Thr, Val) did not increase IL-2 productivity significantly. Furthermore, supplement of the alanine tRNA or threonine tRNA increased IL-2 productivity of IL-2 HL. In summary, we revealed a potential strategy to enhance productivity of recombinant proteins, which may be applied in production of protein drug or design of DNA vaccine.