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E T Peterson

Publications and source records attributed to E T Peterson.

11 recordsLinked to original sources
PubMed1994

In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase.

Small RNAs were selected from a highly degenerate library on the basis of their ability to bind tightly to Escherichia coli phenylalanyl-tRNA synthetase (FRS). The 63 nucleotide library consisted of the acceptor stem and portions of the D and T stems of E. coli tRNA(Phe) flanking a 32 nucleotide randomized region. Because FRS binding relies on a correctly folded tRNA substrate, the selected variants from this library were expected to resemble tRNA(Phe) structure. After seven cycles of selection, the RNA library bound to FRS with similar affinity to that of the E. coli tRNA(Phe), but did not show detectable aminoacylation. Fourteen FRS-specific isolates were sequenced and found to contain an anticodon stem-loop including the anticodon triplet of tRNA(Phe). The tight-binding RNAs fell into two classes depending on the location of this step-loop within the sequence. The acceptor stem defined by the non-randomized sequence was also found to be essential for binding. Mutation of two residues within a common hexanucleotide sequence present in one of the classes reduced binding to FRS. Taken together, these results suggest that in order to bind RNAs tightly, FRS requires the simultaneous interaction of the anticodon stem-loop and acceptor stem, and additional sequences needed for proper folding. This approach should assist in the detection of motifs that resemble tRNA, but are too dissimilar to be identified by sequence comparison.

Base Sequence↗
PubMed1993

Selection for active E. coli tRNA(Phe) variants from a randomized library using two proteins.

In vitro selection was used to isolate active Escherichia coli tRNA(Phe) variants from randomized libraries. Functional tRNAs were first selected by multiple rounds of binding to Escherichia coli phenylalanyl-tRNA synthetase. These variants were then aminoacylated and selected for affinity to elongation factor-Tu. By randomizing potential recognition nucleotides, the importance of residues U20, G34, A35, A36 and U59, previously identified to be required for specific recognition by E. coli phenylalanyl-tRNA synthetase (FRS), was confirmed. However, the sequences of several active variants imply that the wild-type tertiary interactions G10-C25-U45 and A26-G44 are not required for recognition, as previously suggested. Selection of functional tRNAs from a second library randomized at positions normally involved in conserved tertiary interactions revealed new combinations of nucleotides at these positions, suggesting the presence of novel tertiary interactions. In both libraries, active sequences containing deletions were isolated. Taken together, it is clear that FRS is active with substrates having an unexpectedly broad sequence diversity. Finally, the potency of this method is illustrated by the identification of a second class of variants that was isolated by virtue of the presence of an impurity in the FRS preparation.

Base Sequence↗
PubMed1992

Determination of recognition nucleotides for Escherichia coli phenylalanyl-tRNA synthetase.

The nucleotides in Escherichia coli tRNA(Phe) required for recognition by its cognate synthetase have been determined in vitro by measuring the kinetic parameters for aminoacylation using mutant tRNA(Phe) transcripts with purified E. coli tRNA(Phe) synthetase. The substitution of 11 nucleotides in E. coli tRNA(Phe) is shown to decrease the kcat/KM by as much as 1000-fold relative to the wild type. The most important recognition elements are the three anticodon nucleotides G34, A35, and A36. The recognition set also includes nucleotides in the variable pocket (U20 and U59), the acceptor end (A73), and the tRNA central core (G10, C25, A26, G44, and U45). Many of the recognition nucleotides are also among the residues comprising the identity set determined in vivo using an amber suppressor tRNA(Phe) [McClain, W. H., & Foss, K. (1988) J. Mol. Biol. 202, 697-709]. As could be anticipated from the very different methods used, some nucleotides in the identity set determined by the suppressor method were not among the recognition nucleotides and vice versa. The E. coli tRNA(Phe) recognition data can also be compared to the recognition sets for yeast and human tRNA(Phe) determined previously. The results indicate that the mechanism by which phenylalanyl-tRNA synthetases recognize their substrates seems to have diverged somewhat among different species. For example, nucleotide 20 in the D-loop, the anticodon nucleotides and the discriminator base 73 are important for the recognition by all three enzymes. However, recognition of the tRNA central core nucleotides is unique to E. coli FRS.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1992

Recognition nucleotides for human phenylalanyl-tRNA synthetase.

The specificity of the interaction between tRNAPhe and phenylalanyl-tRNA synthetase isolated from human placenta was investigated. Using yeast tRNAPhe transcripts with different point mutations it was shown that all the five recognition points for the yeast phenylalanyl-tRNA synthetase (G20, G34, A35, A36 and A73) are also important for the reaction catalyzed by the human enzyme. A set of mutations in nucleotides involved in tertiary interactions of tRNAPhe revealed that mutations which maintained the proper folding of the molecule had almost no influence on the efficiency of aminoacylation. The most striking difference between the yeast and human phenylalanyl-tRNA synthetases involved a mutation in the lower two base pairs of the anticodon stem. This mutation did not affect aminoacylation with the yeast enzyme, but greatly reduced activity with human phenylalanyl-tRNA synthetase.

Anticodon↗
PubMed1967

Resistance of vaccinated mice to typical and atypical strains of Coccidioides immitis.

In earlier reports, it was shown that mice and monkeys could be immunized against otherwise lethal challenge doses of Coccidioides immitis arthrospores. The vaccine was composed of Formalin-killed, in vitro grown, endosporulating spherules of C. immitis strain Silveira. In this study, mice were immunized as in the earlier work and then challenged intranasally with arthrospores from seven heterologous strains of C. immitis. Two of these strains were typical of the species, and five were atypical with respect to their cultural characteristics and morphology of microscopic structures. The vaccinated animals were well protected against challenge doses that were lethal to a majority of the control animals, regardless of the strain of fungus employed. The infection ratios among surviving vaccinated and control animals were comparable, but demonstrable lesions were generally smaller and less numerous in the vaccinated groups. It is suggested that these strains are at least immunogenically similar, although not necessarily identical, and that a vaccine prepared from a single strain of C. immitis would be practical for an immunization program.

Animals↗