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K Foss

Publications and source records attributed to K Foss.

16 recordsLinked to original sources
PubMed1991

Four sites in the acceptor helix and one site in the variable pocket of tRNA(Ala) determine the molecule's acceptor identity.

The structural features that determine tRNA(Ala) acceptor identity have been studied with amber-suppressor tRNAs in Escherichia coli cells. Previous work established that a wobble pair composed of guanosine at position 3 and uridine at position 70 (G3-U70) in the acceptor helix of tRNA(Ala) is a determinant of the molecule's acceptor identity. We show that additional determinants are located at three other sites in the acceptor helix and at one site in the variable pocket of tRNA(Ala). These latter determinants are less important than G3.U70 since their individual alterations in mutants of tRNA(Ala) have smaller degrading effects on the functions of the molecules, and subsets of the determinants, when combined with G3.U70, are sufficient to switch the identities of several other tRNAs to that of tRNA(Ala). Other workers are using fragments of the tRNA(Ala) acceptor helix to study the molecule's acceptor identity. Our demonstration that the variable pocket contributes to tRNA(Ala) acceptor identity means that such fragments do not faithfully replicate the structure-function relationship of the cellular process.

Anticodon↗
PubMed1991

Rapid determination of nucleotides that define tRNA(Gly) acceptor identity.

Expression of the genetic code depends on the recognition of specific tRNAs by the enzymes that aminoacylate them. A computer comparison of tRNA sequences coupled with analysis of mutant nonsense-suppressor tRNAs has revealed the structural features that distinguish the acceptor identity of Escherichia coli tRNA(Gly) from tRNAs that accept phenylalanine, arginine, lysine, and glutamine. On replacement of several nucleotides in the acceptor stem and anticodon of the latter tRNAs with tRNA(Gly)-derived residues, the resulting molecules acquired a tRNA(Gly) identity.

Anticodon↗
PubMed1990

Nucleotides that determine Escherichia coli tRNA(Arg) and tRNA(Lys) acceptor identities revealed by analyses of mutant opal and amber suppressor tRNAs.

We have constructed an opal suppressor system in Escherichia coli to complement an existing amber suppressor system to study the structural basis of tRNA acceptor identity, particularly the role of middle anticodon nucleotide at position 35. The opal suppressor tRNA contains a UCA anticodon and the mRNA of the suppressed protein (which is easily purified and sequenced) contains a UGA nonsense triplet. Opal suppressor tRNAs of two tRNA(Arg) isoacceptor sequences each gave arginine in the suppressed protein, while the corresponding amber suppressors with U35 in their CUA anticodons each gave arginine plus a second amino acid in the suppressed protein. Since C35 but not U35 is present in the anticodon of wild-type tRNA(Arg) molecules, while the first anticodon position contains either C34 or U34, these results establish that C35 contributes to tRNA(Arg) acceptor identity. Initial characterizations of opal suppressor tRNA(Arg) mutants by suppression efficiency measurements suggest that the fourth nucleotide from the 3' end of tRNA(Arg) (A73 or G73 in different isoacceptors) also contributes to tRNA(Arg) acceptor identity. Wild-type and mutant versions of opal and amber tRNA(Lys) suppressors were examined, revealing that U35 and A73 are important determinants of tRNA(Lys) acceptor identity. Several possibilities are discussed for the general significance of having tRNA acceptor identity in the same positions in different tRNA acceptor types, as exemplified by positions 35 and 73 in tRNA(Arg) and tRNA(Lys).

Anticodon↗
PubMed1988

Association of transfer RNA acceptor identity with a helical irregularity.

The aminoacylation specificity ("acceptor identity") of transfer RNAs (tRNAs) has previously been associated with the position of particular nucleotides, as opposed to distinctive elements of three-dimensional structure. The contribution of a G.U wobble pair in the acceptor helix of tRNA(Ala) to acceptor identity was examined with synthetic amber suppressor tRNAs in Escherichia coli. The acceptor identity was not affected by replacing the G.U wobble pair in tRNA(Ala) with a G.A, C.A, or U.U wobble pair. Furthermore, a tRNA(Ala) acceptor identity was conferred on tRNA(Lys) when the same site in the acceptor helix was replaced with any of several wobble pairs. Additional data with tRNA(Ala) show that a substantial acceptor identity was retained when the G.U wobble pair was translocated to another site in the acceptor helix. These results suggest that the G.U wobble pair induces an irregularity in the acceptor helix of tRNA(Ala) to match a complementary structure in the aminoacylating enzyme.

Base Composition↗
PubMed1988

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket".

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.

DNA Mutational Analysis↗
PubMed1988

Nucleotides that contribute to the identity of Escherichia coli tRNA(Phe).

A series of sequence variants of amber suppressor genes of tRNA(Phe) were synthesized in vitro and cloned in Escherichia coli to examine the contributions of individual nucleotides to identity for amino acid acceptance. Three different but complementary types of tRNA variants were constructed. The first involved the substitution of base-pairs on the cloverleaf stem regions of the E. coli tRNA(Phe). The second type of variant involved total gene synthesis based on wild-type tRNA(Phe) sequences found in Bacillus subtilis and in Halobacterium volcanii. In the third type of variant, the identity of E. coli tRNALys was changed to that of tRNA(Phe). The nucleotides which are important for tRNA(Phe) identity in E. coli are located on the corner of the L-shaped tRNA molecule, where the dihydrouridine loop interacts with the T loop, and extend to the interior opening of the anticodon stem and the adjoining variable loop. The nucleotide sequence on the dihydrouridine stem region, which joins the corner and stem regions, was not successfully studied though it may contribute to tRNA(Phe) identity. The fourth nucleotide from the 3' end of tRNA(Phe) has some importance for identity.

Amino Acid Sequence↗
PubMed1988

Changing the identity of a tRNA by introducing a G-U wobble pair near the 3' acceptor end.

Although the genetic code for protein was established in the 1960's, the basis for amino acid identity of transfer RNA (tRNA) has remained unknown. To investigate the identity of a tRNA, the nucleotides at three computer-identified positions in tRNAPhe (phenylalanine tRNA) were replaced with the corresponding nucleotides from tRNAAla (alanine tRNA). The identity of the resulting tRNA, when examined as an amber suppressor in Escherichia coli, was that of tRNAAla.

Alanine↗
PubMed1987

Suppressor and novel mutants of bacteriophage T4 tRNA(Gly).

We have isolated a weak UGA suppressor of phage T4 tRNA(Gly) in which the anticodon is changed from UCC to UCA. Two secondary mutants lacking suppressor activity are atypical in accumulating tRNA(Gly). Both mutations change the T stem of the cloverleaf model. One involved a G to A change at the 5' base position of the middle base-pair; the second involves a C to U change at a constant base position next to the T loop. The precursor RNAs of the mutants were cleaved in vitro with the catalytic RNA subunit of RNase P. Relative to normal precursor RNA, the precursor mutated at the middle base-pair position of the T stem was cleaved more rapidly, whereas the precursor mutated at the base-pair position next to the T loop was cleaved more slowly.

Base Sequence↗
PubMed1987

Rapid site-specific mutagenesis in plasmids.

A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.

Base Sequence↗
PubMed1984

Hybrid transfer RNA genes in phage T4.

We describe the isolation and characterization of two unusual amber suppressor forms of T4 tRNALeu. The sequences of the suppressor tRNAs can be described as hybrids of wild-type tRNALeu and suppressor tRNAGln molecules: the chain lengths and majority of the nucleotide residues corresponded to tRNALeu, but CUA anticodons flanked by 2-14 residues were identical to tRNAGln. The uncertainty as to the exact number of flanking residues correlated with tRNAGln is due to the similarity of the two tRNA sequences in this region. No evidence was found for changes in other T4 tRNAs. We propose that genes for the hybrid tRNAs were produced by mispairing of DNAs at anticodon segments of tRNALeu and tRNAGln with a double crossover flanking those segments.

Base Sequence↗
PubMed1975

A mutant of escherichia coli defective in removing 3' terminal nucleotides from some transfer RNA precursor molecules.

The conversion of precursor RNA into bacteriophage T4 proline and serine transfer RNAs includes two steps for the enzymatic removal of nucleotides from the 3' ends of RNA chains. Neither of these steps occur following infection of a mutant of Escherichia coli that was previously shown to block the suppressor function of T4 serine transfer RNA. Cell-free extracts of this mutant are furthermore deficient in a wild type enzyme activity that removes nucleotides from the 3' ends of one of the RNA chains described above. The relation of this enzyme to other 3' ribonucleases is not known. We subsequently examined the mutant for its ability to support the biosynthesis of other bacteriophage transfer RNAs. In one instance that is analogous to the proline-serine precursor RNA, maturation of the precursor RNA was blocked during infection of mutant cells. In another instance, precursor RNA maturation was normal, even though this involved the removal of 3'nucleotides. These observations point to the possible existence of at least two 3' ribonucleases for the biosynthesis of transfer RNAs.

Base Sequence↗