Biomedical subjects

W Tate

Publications and source records attributed to W Tate.

8 recordsLinked to original sources

PubMed2000

Release factors and their role as decoding proteins: specificity and fidelity for termination of protein synthesis.

The decoding of stop signals in mRNA requires protein release factors. Two classes of factor are found in both prokaryotes and eukaryotes, a decoding factor and a stimulatory recycling factor. These factors form complexes at the active centre of the ribosome and mimic in overall shape the complexes found at other stages of protein synthesis. The decoding release factor is shaped like a tRNA and has a domain for codon recognition at the decoding site of the ribosome, and a domain for peptidyl-tRNA hydrolysis that is inferred to be near the peptidyltransferase centre. Initial interaction of the decoding factor with the ribosome is a low fidelity event involving multiple contacts with the ribosomal components. A subsequent discrimination step, at present poorly defined, ensures high fidelity of codon recognition.

Animals↗

PubMed1995

Krox20 may play a key role in the stabilization of long-term potentiation.

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 required stimulation sufficient to induce LTP and was prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increased within 20 min of tetanization, was maximal between 1 and 8 h, and was still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast with the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 was of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation.

Animals↗

PubMed1994

Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli.

The termination of protein synthesis in bacteria requires two codon-specific polypeptide release factors, RF-1 and RF-2. A third factor, RF-3, which stimulates the RF-1 and RF-2 activities, was originally identified in Escherichia coli, but it has received little attention since the 1970s. To search for the gene encoding RF-3, we selected nonsense-suppressor mutations by random insertion mutagenesis on the assumption that a loss of function of RF-3 would lead to misreading of stop signals. One of these mutations, named tos-1 (for transposon-induced opal suppressor), mapped to the 99.2 min region on the E. coli chromosome and suppressed all three stop codons. Complementation studies and analyses of the DNA and protein sequences revealed that the tos gene encodes a 59,442-Da protein, with sequence homology to elongation factor EF-G, including G-domain motifs, and that the tos-1 insertion eliminated the C-terminal one-fifth of the protein. Extracts containing the overproduced Tos protein markedly increased the formation of ribosomal termination complexes and stimulated the RF-1 or RF-2 activity in the codon-dependent in vitro termination assay. The stimulation was significantly reduced by GTP, GDP, and the beta,gamma-methylene analog of GTP, but not by GMP. These results fit perfectly with those described in the original publications on RF-3, and the tos gene has therefore been designated prfC. A completely null prfC mutation made by reverse genetics affected the cell growth under the limited set of physiological and strain conditions.

Amino Acid Sequence↗

PubMed1992

Unexpected intron location in non-vertebrate globin genes.

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.

Amino Acid Sequence↗

PubMed1990

Codon recognition in polypeptide chain termination: site directed crosslinking of termination codon to Escherichia coli release factor 2.

An RNA synthesized in vitro was positioned on the Escherichia coli ribosome at the P site with tRNAala, and with a termination codon, UAA, as the next codon in the A site. Such a complex bound stoichiometric amounts of release factor 2 (RF-2); a corresponding RNA with UAC in place of UAA was not a template for the factor. An RNA containing 4-thio-UAA in place of the UAA supported binding of RF-2, and this has allowed site-directed crosslinking from the first position of the termination codon to answer two long standing questions about the termination of protein biosynthesis, the position of the termination codon and its proximity to the release factor during codon recognition. An RF-2.mRNA crosslinked product was detected, indicating the release factor and the termination codon are in close physical contact during the codon recognition event of termination. The 4-thio-U crosslinked also to the ribosome but only to the 30S subunit, and the proteins and the rRNA site concerned were identified. RF-2 decreased significantly the crosslinking to the ribosomal components, but no new crosslink sites were found. If the stop codon was deliberately displaced from the decoding site by one codon's length then a different pattern of crosslinking in particular to the rRNA resulted. These observations are consistent with a model of codon recognition by RF-2 at the decoding site, without a major shift in position of the codon.

Base Sequence↗

PubMed1990

Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes.

A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence. High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes. This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon. Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible. A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency. The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone. This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting.

Base Sequence↗

PubMed1984

Cloning of the Escherichia coli release factor 2 gene.

The protein release factor 2 (RF2) participates in Escherichia coli polypeptide chain termination with codon specificity (UAA or UGA). A colicin E1 recombinant identified in the Carbon and Clarke E. coli bank contains the protein release factor 2 gene. A 1.7-kilobase E. coli fragment has been subcloned into the plasmid pUC9 vector. Bacterial cells, containing the plasmid recombinant, produce elevated levels of protein release factor 2 as detected by an immune precipitation assay and in vitro measurement of UGA-directed peptide chain termination and [3H]UGA codon recognition.

Bacteriocin Plasmids↗

PubMed1977

Characterization of reticulocyte release factor.

The release factor (RF) of reticulocytes has been purified to greater than 75% homogeneity. The RF has a native molecular weight of 105,000 and subunit molecular weight of 56,500. The RF protein will bind to reticulocyte ribosomes in response to UAAA, UAGA, or UGAA and therefore participates in codon recognition. The fraction possess a ribosome-dependent GTPase activity. The RF is stimulated in its activity by a second protein fraction.

Animals↗