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Y M Hou

Publications and source records attributed to Y M Hou.

49 records · Page 3Linked to original sources
PubMed1992

Novel transfer RNAs that are active in Escherichia coli.

Many of the mammalian mitochondrial tRNAs contain significant nucleotide deletions in the dihydrouridine (D) stem or T psi C stem, so that they cannot fold into the canonical cloverleaf structure. This suggests that alternative forms and shapes are possible for a mitochondrial tRNA that functions in the specialized translational apparatus of the mammalian mitochondria. The question of whether significant structural alterations may be accommodated by a bacterial protein synthesis machinery, such as in Escherichia coli, is unanswered. In this work, all but ten positions in the gene for the 76-nucleotide coding sequence of an E. coli amber suppressor tRNA were permuted and screened for biological activity in vivo. Sequence analysis of a collection of biologically active variants established that many have unusual structures that include base-pair mismatches in helical stems, substitutions of normally conserved bases, and deletions. Independent mutations were obtained that weaken base pairs or tertiary interactions that normally stabilize the coaxial stacking of the D and anticodon stems, suggesting that the translational apparatus can accommodate considerable flexibility in this part of the molecule. The results demonstrate the capacity of the bacterial protein synthetic apparatus to accommodate altered tRNA structures that are not represented by any naturally occurring tRNAs.

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↗
PubMed1991

Sequence determination and modeling of structural motifs for the smallest monomeric aminoacyl-tRNA synthetase.

Polypeptide chains of 19 previously studied Escherichia coli aminoacyl-tRNA synthetases are as large as 951 amino acids and, depending on the enzyme, have quaternary structures of alpha, alpha 2, alpha 2 beta 2, and alpha 4. These enzymes have been organized into two classes which are defined by sequence motifs that are associated with specific three-dimensional structures. We isolated, cloned, and sequenced the previously uncharacterized gene for E. coli cysteine-tRNA synthetase (EC 6.1.1.16) and showed that it encodes a protein of 461 amino acids. Biochemical analysis established that the protein is a monomer, thus establishing this enzyme as the smallest known monomeric synthetase. The sequence shows that cysteine-tRNA synthetase is a class I enzyme that is most closely related to a subgroup that includes the much larger methionine-, isoleucine-, leucine-, and valine-tRNA synthetases, which range in size from 677 to 951 amino acids. The amino-terminal 293 amino acids of the cysteine enzyme can be modeled as a nucleotide-binding fold that is more compact than that of its closest relatives by virtue of truncations of two insertions that split the fold. This smaller nucleotide-binding fold accounts for much of the reduced size of the cysteine enzyme and establishes the limit to which the structure of this domain is contracted in the five members of this subgroup of class I enzymes.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of D-xylose isomerase gene from Escherichia coli.

Gene coding for E. coli D-xylose isomerase was cloned into the plasmid pWR13 from the plasmid pXO100 containing D-xylose operon. Several subclones harboring DNA fragments of different size obtained by step- by-step shortening with Bal31 nuclease were directly sequenced. The complete nucleotide sequence of the 1.6kb DNA fragment carrying D-xylose isomerase gene was determined. The structural gene of 1320 nucleotides codes for a protein of 440 amino acids. The additional 209 nucleotides at 5' end and 82 nucleotides at 3' end include Shine-Dalgarno ribosome-binding site and the transcriptional termination codon, respectively.

Aldose-Ketose Isomerases↗
PubMed1989

Evidence that a major determinant for the identity of a transfer RNA is conserved in evolution.

We observed recently that a single G3.U70 base pair in the amino acid acceptor stem of an Escherichia coli alanine tRNA is a major determinant for its identity. Inspection of tRNA sequences shows that G3.U70 is unique to alanine in E. coli and is present in eucaryotic cytoplasmic alanine tRNAs. We show here that single nucleotide changes of G3.U70 to A3.U70 or to G3.C70 eliminate in vitro aminoacylation of an insect and of a human alanine tRNA by the respective homologous synthetase. Compared to the influence of G3.U70, other sequence variations in tRNAAla have a relatively small effect on aminoacylation by the insect and human enzymes. In addition, while these eucaryotic tRNAs have nucleotide differences from E. coli alanine tRNA, they are heterologously charged only with alanine when expressed in E. coli. The results indicate a functional role for G3.U70 that is conserved in evolution. They also suggest that the sequence differences between E. coli and the eucaryotic alanine tRNAs at sites other than the conserved G3.U70 do not create major determinants for recognition by any other bacterial enzyme.

Alanine-tRNA Ligase↗
PubMed1989

Modeling with in vitro kinetic parameters for the elaboration of transfer RNA identity in vivo.

A tRNA with "double identity" was created, and this tRNA was demonstrated in vitro to aminoacylate quantitatively with either of two amino acids. In contrast, acceptance of only one of these amino acids was observed in vivo, and a simple manipulation determined which one was accepted. Kinetic parameters were obtained for aminoacylation with each amino acid of the tRNA with double identity and of related tRNAs. Modeling with these parameters largely explains which amino acid specificity is observed in vivo. The results delineate some of the kinetic boundaries for the design and accommodation of tRNA sequence variations in the elaboration of identity in vivo.

Alanine↗
PubMed1989

A single base pair affects binding and catalytic parameters in the molecular recognition of a transfer RNA.

A single G3.U70 base pair in the acceptor helix is a major determinant of the identity of an alanine transfer RNA. Alteration of this base pair to A.U or G.C prevents aminoacylation with alanine. We show here that, at approximate physiological conditions (pH 7.5, 37 degrees C), high concentrations of the mutant A3.U70 species do not inhibit aminoacylation of a wild-type alanine tRNA. The observation suggests that, under these conditions, the G3 to A3 substitution increases Km for tRNA by more than 30-fold. Other experiments at pH 7.5 show that no aminoacylation of A3.U70, G3.C70, or U3.G70 mutant tRNAs occurs with substrate levels of enzyme. This suggests that kcat for these mutant tRNAs is sharply reduced as well and that the catalytic defect is not due to slow release of charged mutant tRNAs from the enzyme. Investigations were also done at pH 5.5, where association of tRNAs with synthetases is generally stronger and where binding can be conveniently measured apart from aminoacylation. Under these conditions, the binding of the A3.U70 and G3.C70 species is readily detected and is only 3-5-fold weaker than the binding of the wild-type tRNA. Although the A3.U70 species was demonstrated to compete with the wild-type tRNA for the same site on the enzyme, no aminoacylation could be detected. Thus, even when conditions are adjusted to obtain strong competitive binding, a sharp reduction in kcat prevents aminoacylation of a tRNA(Ala) species with a substitution at position 3.70.

Base Composition↗
PubMed1989

Molecular dissection of a transfer RNA and the basis for its identity.

The recognition of transfer RNAs (tRNAs) by aminoacyl tRNA synthetases establishes the connection between amino acids and trinucleotides. However, for E. coli alanine tRNA the trinucleotide sequence which specifies alanine is not important for recognition. Instead a single base pair is a major determinant for the identity of this tRNA. Even a synthetic RNA microhelix with seven base pairs can be aminoacylated if it includes the major determinant.

Alanine↗
PubMed1988

Expression of the mouse metallothionein-I gene in Escherichia coli: increased tolerance to heavy metals.

The cDNA of mouse metallothionein, a small metal-binding protein rich in cysteine, has been cloned downstream from a bacterial inducible promoter and expressed in Escherichia coli. Upon induction, E. coli harboring this cDNA clone contained a protein species readily labelled by [35S]cysteine in vivo and incorporated 10-times as much 109Cd from the medium than would otherwise be the case. We show that expression of metallothionein endows resistance in E. coli to heavy metal ions such as mercury, silver, copper, cadmium and zinc by sequestering rather than exclusion or conversion, common mechanisms of metal resistance in bacteria.

Animals↗
PubMed1988

A simple structural feature is a major determinant of the identity of a transfer RNA.

Analysis of a series of mutants of an Escherichia coli alanine transfer RNA shows that substitution of a single G-U base pair in the acceptor helix eliminates aminoacylation with alanine in vivo and in vitro. Introduction of that base pair into the analogous position of a cysteine and a phenylalanine transfer RNA confers upon each the ability to be aminoacylated with alanine. Thus, as little as a single base pair can direct an amino acid to a specific transfer RNA.

Base Composition↗