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At least 109 records · Page 6Linked to original sources
PubMed1999

Efficient 50S ribosome-catalyzed peptide bond synthesis with an aminoacyl minihelix.

RNA minihelices that recreate the amino acid acceptor domain of the two-domain L-shaped tRNA molecule are substrates for specific aminoacylation by tRNA synthetases. Some lines of evidence suggest that this domain arose independently of and predated the second, anticodon-containing domain. With puromycin and a minihelix charged with alanine, we show here efficient 50S ribosome catalyzed peptide synthesis. The aminoacyl minihelix is as active as aminoacyl tRNA in the synthetic reaction. The high efficiency of the charged minihelix is due to a relatively strong interaction with the 50S particle. In contrast, an aminoacyl RNA fragment that recreates the 3'-side of the tRNA acceptor stem has a much weaker interaction with the 50S particle. These results are consistent with the minihelix domain being the major loci for tRNA interactions with the 50S ribosome. They may also have implications for the historical development of RNA-based systems of peptide synthesis.

Acylation↗
PubMed2000

Importance of discriminator base stacking interactions: molecular dynamics analysis of A73 microhelix(Ala) variants.

Transfer of alanine from Escherichia coli alanyl-tRNA synthetase (AlaRS) to RNA minihelices that mimic the amino acid acceptor stem of tRNA(Ala) has been shown, by analysis of variant minihelix aminoacylation activities, to involve a transition state sensitive to changes in the 'discriminator' base at position 73. Solution NMR has indicated that this single-stranded nucleotide is predominantly stacked onto G1 of the first base pair of the alanine acceptor stem helix. We report the activity of a new variant with the adenine at position 73 substituted by its non-polar isostere 4-methylindole (M). Despite lacking N7, this analog is well tolerated by AlaRS. Molecular dynamics (MD) simulations show that the M substitution improves position 73 base stacking over G1, as measured by a stacking lifetime analysis. Additional MD simulations of wild-type microhelix(Ala) and six variants reveal a positive correlation between N73 base stacking propensity over G1 and aminoacylation activity. For the two DeltaN7 variants simulated we found that the propensity to stack over G1 was similar to the analogous variants that contain N7 and we conclude that the decrease in aminoacylation efficiency observed upon deletion of N7 is likely due to loss of a direct stabilizing interaction with the synthetase.

Acylation↗
PubMed1999

Identification of discriminator base atomic groups that modulate the alanine aminoacylation reaction.

Specific aminoacylation of tRNAs involves activation of an amino acid with ATP followed by amino acid transfer to the tRNA. Previous work showed that the transfer of alanine from Escherichia coli alanyl-tRNA synthetase to a cognate RNA minihelix involves a transition state sensitive to changes in the tRNA acceptor stem. Specifically, the "discriminator" base at position 73 of minihelix(Ala) is a critical determinant of the transfer step of aminoacylation. This single-stranded nucleotide has previously been shown by solution NMR to be stacked predominantly onto G(1) of the first base pair of the alanine acceptor stem helix. In this work, RNA duplex(Ala) variants were prepared to investigate the role of specific discriminator base atomic groups in aminoacylation catalytic efficiency. Results indicate that the purine structure appears to be important for stabilization of the transition state and that major groove elements are more critical than those located in the minor groove. This result is in accordance with the predicted orientation of a class II synthetase at the end of the acceptor helix. In particular, substitution of the exocyclic amino group of A(73) with a keto-oxygen resulted in negative discrimination at this site. Taken together, these new results are consistent with the involvement of major groove atomic groups of the discriminator base in the formation of the transition state for the amino acid transfer step.

Acylation↗
PubMed1994

Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate.

The class II Escherichia coli alanine tRNA synthetase aminoacylates RNA miniduplexes, which reconstruct the acceptor end of alanine tRNA with the critical G3:U70 base pair. A benzophenone photoaffinity label attached adjacent to G3:U70 in a miniduplex substrate was previously cross-linked to a long enzyme peptide that begins at Gly161 between the class-defining motifs 2 and 3 [Musier-Forsyth, K., & Schimmel, P. (1994) Biochemistry 33, 773-779]. To identify side chains in this peptide that potentially contribute hydrogen bonding or catalytic determinants for the RNA-dependent step of the aminoacylation reaction, peptide functional side chains that are conserved among sequenced alanine enzymes (Asp, Asn, Arg, Glu, Gln, and Tyr) were individually replaced. Of the 21 mutant proteins so generated, one was identified that was not viable even though it accumulated in vivo. This Asp235-->Ala mutant enzyme is defective in the rate of transfer of the activated amino acid to the 3'-end of the RNA substrate. The conserved Asp235 is at the beginning of motif 3. By comparison with the crystal structure of the related class II yeast aspartate tRNA synthetase complexed with tRNA(Asp) (Cavarelli et al., 1993), we suggest that D235 is not in direct contact with acceptor helix base pairs such as G3:U70. Instead, we propose that D235 contributes to transfer-step interactions at the 3'-end of alanine tRNA. Because D235 in alanine tRNA synthetase is at the beginning of one of the conserved motifs that define class II tRNA synthetases, this region of the structure may in general be important for the transfer step.

Alanine-tRNA Ligase↗
PubMed1992

Functional contacts of a transfer RNA synthetase with 2'-hydroxyl groups in the RNA minor groove.

The functional analysis of determinants on RNA has been largely limited to molecules that contain naturally occurring ribonucleotides, so little is known about the role of 2'-hydroxyl groups in protein-RNA recognition. A single base pair (G3.U70) in the acceptor stem of tRNA(Ala) is the principal element for specific recognition by Escherichia coli alanine-tRNA synthetase. This tRNA synthetase aminoacylates small RNA helices that contain the G3.U70 base pair. Furthermore, removal of the G3 exocyclic 2-amino group that projects into the minor groove eliminates aminoacylation. This 2-amino group is flanked on either side by ribose 2'-hydroxyl groups that line the minor groove. Here we use chemical synthesis to construct 32 helices that make deoxy and O-methyl substitutions of individual and multiple 2'-hydroxyl groups near and beyond the G3.U70 base pair and find that functional 2'-hydroxyl contacts are clustered within a few ångstroms of the critical 2-amino group. These contacts are highly specific and make a thermodynamically significant contribution to RNA recognition.

Alanine-tRNA Ligase↗
PubMed1995

Permutation of a pair of tertiary nucleotides in a transfer RNA.

The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human.

Anticodon↗
PubMed2002

Modulation of tRNAAla identity by inorganic pyrophosphatase.

A highly sensitive assay of tRNA aminoacylation was developed that directly measures the fraction of aminoacylated tRNA by following amino acid attachment to the 3'-(32)P-labeled tRNA. When applied to Escherichia coli alanyl-tRNA synthetase, the assay allowed accurate measurement of aminoacylation of the most deleterious mutants of tRNA(Ala). The effect of tRNA(Ala) identity mutations on both aminoacylation efficiency (k(cat)/K(M)) and steady-state level of aminoacyl-tRNA was evaluated in the absence and presence of inorganic pyrophosphatase and elongation factor Tu. Significant levels of aminoacylation were achieved for tRNA mutants even when the k(cat)/K(M) value is reduced by as much as several thousandfold. These results partially reconcile the discrepancy between in vivo and in vitro analysis of tRNA(Ala) identity.

Escherichia coli↗
PubMed1990

Modification of the anticodon wobble position of tRNA(Ala) in vitro does not require 5' or 3' processing.

Maturation of eukaryotic tRNA molecules requires nuclear processing as well as nuclear and cytoplasmic modification of specific nucleotides. Nucleotide modifications within the anticodon are found in the majority of all tRNAs and are among the last maturation events to occur in vivo. We show that 5' and 3' processing of SP6 polymerase-generated transcripts are not necessary for the in vitro modification of A----I in the anticodon of tRNA(Ala).

Animals↗
PubMed1991

Further improvement in protecting method for the oligonucleotide synthesis in terms of a cellulose acetate derivative as a polymer support.

For further improvement in the investigation to utilize a cellulose acetate derivative as a novel type of polymer-support for the synthesis of oligonucleotides, the investigations on utilizing another spacer; on protecting groups for O6-position of guanosine unit, ribothymidine, and pseudouridine; and on a novel protecting group for the introduction of phosphate function at 5'-terminal position, targeting the syntheses of 13-mer, ApApGpGpApApApApUpUpApUpG, 11-mer, pCpUpCpGpUpCpCpApCpCpA, and 12-mer, UpCpCpGpGprTp- psipCpGpApUpU, found in the partial structures of a yeast tRNA(Ala), will be described in detail.

Base Sequence↗
PubMed1991

Mutant aminoacyl-tRNA synthetase that compensates for a mutation in the major identity determinant of its tRNA.

A single G3.U70 base pair in the acceptor helix is the major determinant for the identity of alanine transfer RNAs (Hou & Schimmel, 1988). Introduction of this base pair into foreign tRNA sequences confers alanine acceptance on them. Moreover, small RNA helices with as few as seven base pairs can be aminoacylated with alanine, provided that they encode the critical base pair (Francklyn & Schimmel, 1989). Alteration of G3.U70 to G3.C70 abolishes aminoacylation with alanine in vivo and in vitro. We describe here the mutagenesis and selection of a single point mutation in Escherichia coli Ala-tRNA synthetase that compensates for a G3.C70 mutation in tRNAAla. The mutation maps to a region previously implicated as proximal to the acceptor end of the bound tRNA. In contrast to the wild-type enzyme, the mutant charges small RNA helices that encode a G3.C70 base pair. However, the mutant enzyme retains specificity for alanine tRNA and can serve as the sole source of Ala-tRNA synthetase in vivo. The results demonstrate the capacity of an aminoacyl-tRNA synthetase to compensate through a single amino acid substitution for mutations in the major determinant of its cognate tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1996

An alanine tRNA gene cluster from Nephila clavipes.

We report the sequence of a 2.3-kb genomic DNA fragment from the orb-web spider, Nephila clavipes (Nc). The fragment contains four regions of high homology to tRNA(Ala). The members of this irregularly spaced cluster of genes are oriented in the same direction and have the same anticodon (GCA), but their sequence differs at several positions. Initiation and termination signals, as well as consensus intragenic promoter sequences characteristic of tRNA genes, have been identified in all genes. tRNA(Ala) are involved in the regulation of the fibroin synthesis in the large ampullate Nc glands.

Animals↗
PubMed1998

Structural and dynamic helix geometry alterations induced by mismatch base pairs in double-helical RNA.

A ribooligonucleotide microhelix derived from the acceptor stem of Escherichia coli tRNA(Ala) having a C3-A70 mismatch in place of the G3-U70 wobble pair in the wild-type tRNA(Ala), and a sequence variant with a regular U3-A70 base pair have been investigated by NMR. In vivo, suppressor tRNA(Ala) variants with C3-A70 (as well as several other) mismatch pairs are substrates for alanyl-tRNA synthetase (ARS), supporting the hypothesis of an 'indirect' recognition of the identity element 3-70 mismatch pair via structural modifications caused by the mispair in comparison to canonical A-RNA helices. It is demonstrated that the C-A mismatch likewise induces helix geometry alterations, in particular with respect to base stacking in the vicinity of the mismatch. However, with reference to the 'wild-type' G3-U70 microhelix, destacking in the C3-A70 acceptor stem duplex occurs in the opposite direction from the mismatch pair. Therefore it is concluded that the locally enhanced conformational flexibility or dynamics associated with the structural changes induced by the mismatch pairs could be an essential prerequisite for optimal adaptation of the tRNA(Ala) acceptor stem to the contact region of the ARS.

Escherichia coli↗
PubMed1999

A bayesian statistical algorithm for RNA secondary structure prediction.

A Bayesian approach for predicting RNA secondary structure that addresses the following three open issues is described: (1) the need for a representation of the full ensemble of probable structures; (2) the need to specify a fixed set of energy parameters; (3) the desire to make statistical inferences on all variables in the problem. It has recently been shown that Bayesian inference can be employed to relax or eliminate the need to specify the parameters of bioinformatics recursive algorithms and to give a statistical representation of the full ensemble of probable solutions with the incorporation of uncertainty in parameter values. In this paper, we make an initial exploration of these potential advantages of the Bayesian approach. We present a Bayesian algorithm that is based on stacking energy rules but relaxes the need to specify the parameters. The algorithm returns the exact posterior distribution of the number of destabilizing loops, stacking energy matrices, and secondary structures. The algorithm generates statistically representative structures from the full ensemble of probable secondary structures in exact proportion to the posterior probabilities. Once the forward recursions for the algorithm are completed, the backward recursive sampling executes in O(n) time, providing a very efficient approach for generating representative structures. We demonstrate the utility of the Bayesian approach with several tRNA sequences. The potential of the approach for predicting RNA secondary structures and presenting alternative structures is illustrated with applications to the Escherichia coli tRNA(Ala) sequence and the Xenopus laevis oocyte 5S rRNA sequence.

Algorithms↗
PubMed1996

Specific function of a G.U wobble pair from an adjacent helical site in tRNA(Ala) during recognition by alanyl-tRNA synthetase.

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.

Alanine-tRNA Ligase↗
PubMed2006

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I.

Effects of tRNA(Ala)(UGC) and its derivative devoid of the 3'-ACCA motif [tRNA(Ala)(UGC)DeltaACCA] on the cleavage of the ColE1-like plasmid-derived RNA I were analysed in vivo and in vitro. In an amino-acid-starved relA mutant, in which uncharged tRNAs occur in large amounts, three products of specific cleavage of RNA I were observed, in contrast to an otherwise isogenic relA(+) host. Overexpression of tRNA(Ala)(UGC), which under such conditions occurs in Escherichia coli mostly in an uncharged form, induced RNA I cleavage and resulted in an increase in ColE1-like plasmid DNA copy number. Such effects were not observed during overexpression of the 3'-ACCA-truncated tRNA(Ala)(UGC). Moreover, tRNA(Ala)(UGC), but not tRNA(Ala)(UGC)DeltaACCA, caused RNA I cleavage in vitro in the presence of MgCl(2). These results strongly suggest that tRNA-dependent RNA I cleavage occurs in ColE1-like plasmid-bearing E. coli, and demonstrate that tRNA(Ala)(UGC) participates in specific degradation of RNA I in vivo and in vitro. This reaction is dependent on the presence of the 3'-ACCA motif of tRNA(Ala)(UGC).

Bacteriocin Plasmids↗
PubMed1996

Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage.

We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.

Animals↗
PubMed2005

Molecular basis of alanine discrimination in editing site.

AlaX is the homologue of the class II alanyl-tRNA synthetase editing domain and has been shown to exhibit autonomous editing activity against mischarged tRNA(Ala). Here, we present the structures of AlaX from the archaeon Pyrococcus horikoshii in apo form, complexed with zinc, and with noncognate amino acid l-serine and zinc. Together with mutational analysis, we demonstrated that the conserved Thr-30 hydroxyl group located near the beta-methylene of the bound serine is responsible for the discrimination of noncognate serine from cognate alanine, based on their chemical natures. Furthermore, we confirmed that the conserved Gln-584 in alanyl-tRNA synthetase, which corresponds to Thr-30 of AlaX, is also critical for discrimination. These observations strongly suggested conservation of the chemical discrimination among trans- and cis-editing of tRNA(Ala).

Acylation↗