Dataset Information

The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2.

ABSTRACT: Okadaic acid, a tumour promoter which potently inhibits protein phosphatases, inhibited translation in the reticulocyte-lysate cell-free system. Inhibition was dose-dependent, with half-maximal effects occurring at 20-40 nM-okadaic acid. Inhibition of translation by okadaic acid resulted in the accumulation of polyribosomes, indicating that it was due to a decrease in the rate of elongation relative to initiation. Okadaic acid (at concentrations which inhibited translation) caused increased phosphorylation of a number of proteins in the lysate. Prominent among these was a protein of Mr 100,000, which has previously been identified as elongation factor 2 (EF-2). EF-2 is a specific substrate for a Ca2+/calmodulin-dependent protein kinase, which phosphorylates EF-2 on threonine residues. The Mr-100,000 band was phosphorylated exclusively on threonine residues, and its degree of 32P labelling was decreased by the Ca2+ chelator EGTA and by the calmodulin antagonist trifluoperazine. These agents attenuated the effects of okadaic acid on EF-2 phosphorylation and translation. When ranges of concentrations of each agent were tested, their effects on EF-2 labelling correlated well with their ability to reverse the okadaic acid-induced inhibition of translation. These findings demonstrate that increased phosphorylation of EF-2 results in an impairment of peptide-chain elongation when natural mRNA is used. The possible physiological role of EF-2 phosphorylation in the control of translation is discussed.

SUBMITTER: Redpath NT

PROVIDER: S-EPMC1133230 | biostudies-other | 1989 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

Dataset Information

The tumour promoter okadaic acid inhibits reticulocyte-lysate protein synthesis by increasing the net phosphorylation of elongation factor 2.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets