Biomedical subjects

William C Merrick

Publications and source records attributed to William C Merrick.

23 records · Page 2Linked to original sources

PubMed2002

Characterization of mammalian eIF2A and identification of the yeast homolog.

To begin the physical characterization of eukaryotic initiation factor (eIF) 2A, a translation initiation factor that binds Met-tRNA(i), tryptic peptides from rabbit reticulocyte eIF2A were analyzed to obtain amino acid sequence information. Sequences for 8 peptides were matched to three different expressed sequence tag clones. The sequence predicted for eIF2A is 585 amino acids. Matching of the cDNA sequence to the human genome revealed that the eIF2A mRNA is made up of 15 or 16 exons, and the gene is contained on chromosome 3. A homolog in Saccharomyces cerevisiae was identified, YGR054W, which is a non-essential gene. Hemagglutinin-tagged yeast eIF2A localizes on both 40 S and 80 S ribosomes. A knockout of both eIF2A and eIF5B yielded a "synthetically sick" yeast strain with a severe slow growth phenotype. The phenotype of this double mutant and the biochemical localization suggest that eIF2A participates in translation initiation. eIF2A does not appear to participate in re-initiation as the DeltaeIF2A strain shows the same level of GCN4 induction with amino acid starvation as seen in wild type yeast. The lack of any apparent phenotype in the DeltaeIF2A strain suggests that eIF2A functions in a minor pathway, perhaps internal initiation or in the translation of a small number of specific mRNAs.

Amino Acid Sequence↗

PubMed2002

Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40 S ribosomal subunits to the initiation codon of beta-globin mRNA.

p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.

Animals↗

PubMed2002

eIF4A: the godfather of the DEAD box helicases.

eIF4A has long been considered the "gold standard" for DEAD box helicases. In large measure, this reflected two items: first, the role of eIF4A in protein synthesis initiation was relatively well established. Second, a wide variety of biochemical studies had established the ability of eIF4A to bind nucleic acids in an ATP-dependent manner, to hydrolyze ATP in an RNA-dependent manner, and to unwind RNA duplexes in an ATP-dependent manner. In this article, these basic observations are reviewed for biochemical consistency and also interpreted in light of the available crystal structures for DEAD box proteins. The role of non-processive vs. processive helicase activity in protein synthesis is discussed. Also examined is the influence of ancillary protein factors (eIF4B, eIF4G, and eIF4H) on this activity. Finally, the "real" role(s) for eIF4A helicase activity in protein synthesis is discussed and related to other circumstances that likely also involve the use of non-processive or slightly processive DEAD box helicases (ribosome biosynthesis, RNA splicing).

Amino Acid Motifs↗

PubMed2001

Regulation of internal ribosome entry site-mediated translation by eukaryotic initiation factor-2alpha phosphorylation and translation of a small upstream open reading frame.

Adaptation to amino acid deficiency is critical for cell survival. In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2alpha by the kinase GCN2. This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases. Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells. This increase requires both GCN2 phosphorylation of eIF2alpha and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5'-leader of the transporter mRNA. When this 5'-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation. Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5'-leader, in a manner dependent on eIF2alpha phosphorylation and translation of the 48-amino acid uORF. This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2alpha phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.

5' Untranslated Regions↗

PubMed2001

The NS5A protein of bovine viral diarrhoea virus interacts with the alpha subunit of translation elongation factor-1.

A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen. The NS5A interactor was identified as the alpha subunit of bovine translation elongation factor 1A (eEF1A). Cell-free binding studies were performed with chimeric NS5A fused to glutathione S-transferase (GST-NS5A) expressed in bacteria. GST-NS5A bound specifically to both in vitro-translated and mammalian cell-expressed eEF1A. Moreover, purified eEF1A bound specifically to GST-NS5A attached to a solid phase. Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes. NS5A from all BVDV strains tested so far interacted with eEF1A. The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.

Animals↗