The N-Terminus of GalE Induces tmRNA Activity in Escherichia coli

BACKGROUND: The tmRNA quality control system recognizes stalled translation complexes and facilitates ribosome recycling in a process termed 'ribosome rescue'. During ribosome rescue, nascent chains are tagged with the tmRNA-encoded SsrA peptide, which targets tagged proteins for degradation. In Escherichia coli, tmRNA rescues ribosomes arrested on truncated messages, as well as ribosomes that are paused during elongation and termination. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a new translational pausing determinant that leads to SsrA peptide tagging of the E. coli GalE protein (UDP-galactose 4-epimerase). GalE chains are tagged at more than 150 sites, primarily within distinct clusters throughout the C-terminal domain. These tagging sites do not correspond to rare codon clusters and synonymous recoding of the galE gene had little effect on tagging. Moreover, tagging was largely unaffected by perturbations that either stabilize or destabilize the galE transcript. Examination of GalE-thioredoxin (TrxA) fusion proteins showed that the GalE C-terminal domain is no longer tagged when fused to an N-terminal TrxA domain. Conversely, the N-terminus of GalE induced tagging within the fused C-terminal TrxA domain. CONCLUSIONS/SIGNIFICANCE: These findings suggest that translation of the GalE N-terminus induces subsequent tagging of the C-terminal domain. We propose that co-translational maturation of the GalE N-terminal domain influences ribosome pausing and subsequent tmRNA activity

E. coli metabolic protein aldehydealcohol dehydrogenase-E binds to the ribosome: a unique moonlighting action revealed

It is becoming increasingly evident that a high degree of regulation is involved in the protein synthesis machinery entailing more interacting regulatory factors. A multitude of proteins have been identified recently which show regulatory function upon binding to the ribosome. Here, we identify tight association of a metabolic protein aldehyde-alcohol dehydrogenase E (AdhE) with the E. coli 70S ribosome isolated from cell extract under low salt wash conditions. Cryo-EM reconstruction of the ribosome sample allows us to localize its position on the head of the small subunit, near the mRNA entrance. Our study demonstrates substantial RNA unwinding activity of AdhE which can account for the ability of ribosome to translate through downstream of at least certain mRNA helices. Thus far, in E. coli, no ribosome-associated factor has been identified that shows downstream mRNA helicase activity. Additionally, the cryo-EM map reveals interaction of another extracellular protein, outer membrane protein C (OmpC), with the ribosome at the peripheral solvent side of the 50S subunit. Our result also provides important insight into plausible functional role of OmpC upon ribosome binding. Visualization of the ribosome purified directly from the cell lysate unveils for the first time interactions of additional regulatory proteins with the ribosom

Chemoselective palladium-catalyzed cyanation of alkenyl halides

A palladium-catalyzed cyanation of alkenyl halides using acetone cyanohydrin is described. A number of structurally diverse alkenylic nitrile containing compounds was prepared in one step under optimized conditions. The reaction proved to be efficient, chemoselective, easy to perform, and tolerant of a number of functional groups

Mammalian miRNA RISC Recruits CAF1 and PABP to Affect PABP-Dependent Deadenylation

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3′UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation. © 2009 Elsevier Inc. All rights reserved