The Role of Ribosomal Protein L11 in Class I Release Factor-mediated Translation Termination and Translational Accuracy

Abstract

It has been suggested from in vivo and cryoelectron micrographic studies that the large ribosomal subunit protein L11 and its N-terminal domain play an important role in peptide release by, in particular, the class I release factor RF1. In this work, we have studied in vitro the role of L11 in translation termination with ribosomes from a wild type strain (WT-L11), an L11 knocked-out strain (⌬L11), and an L11 N terminus truncated strain (Cter-L11). Our data show 4 -6-fold reductions in termination efficiency (k cat /K m ) of RF1, but not of RF2, on ⌬L11 and Cter-L11 ribosomes compared with wild type. There is, at the same time, no effect of these L11 alterations on the maximal rate of ester bond cleavage by either RF1 or RF2. The rates of dissociation of RF2 but not of RF1 from the ribosome after peptide release are somewhat reduced by the L11 changes irrespective of the presence of RF3, and they cause a 2-fold decrease in the missense error. Our results suggest that the L11 modifications increase nonsense suppression at UAG codons because of the reduced termination efficiency of RF1 and that they decrease nonsense suppression at UGA codons because of a decreased missense error level.L11 is a highly conserved ribosomal 14.8-kDa protein located at the base of the L7/L12 stalk of the ribosome, which is essential for several steps in protein synthesis (1-5). L11 binds to the nucleotides 1051-1108 of Escherichia coli 23 S rRNA, commonly called the L11 binding region (L11BR) 2 (6), which constitutes the GTPase-associated center, an important sector of the bacterial ribosome, where all of the translational GTPases bind and hydrolyze GTP in the course of their action (7). This is also the site of action for the thiazole antibiotics thiostrepton and micrococcin (8 -10). In addition to the GTPases, some other translational factors interact with L11 and the L11BR in functionally important ways. The class I release factors RF1 and RF2 belong to this group. RF1 recognizes the stop codons UAG and UAA, whereas RF2 recognizes the stop codons UGA and UAA in the A site of the ribosome (11). Recent cryo-EM studies with termination complexes containing RF2 (12, 13) and RF1 3 show that these two factors acquire very similar overall conformations on the ribosome. Although they bind to the decoding center on the 30 S subunit and reach up to the peptidyltransferase center on the 50 S subunit to induce release of the nascent peptide chain, they interact with L11BR (12, 13) and L11 3 with their flexible domain I. These observations are in line with previous suggestions, based on biochemical and genetic experiments, that there are interactions between L11 and the release factors (4, 14 -18).The L11 protein consists of two domains, a tightly folded N-terminal domain (NTD), which is loosely connected to the large compact C-terminal domain (CTD). The CTD of L11 is in stable contact with the L11BR RNA, whereas the NTD can change its position and proximity with respect to the rest of the protein (19). Earlier biochemical and genetic studies i...

Selenocysteine Insertion at a Predefined UAG Codon in a Release Factor 1 (RF1)-depleted Escherichia coli Host Strain Bypasses Species Barriers in Recombinant Selenoprotein Translation

“…4, red arrow), hence produced although RF1 was not present. Perhaps this truncation was RF2-mediated, especially as stoichiometry between ribosomal protein L11, RF1 and RF2, known to affect termination efficiency (43), is considerably distorted in these cells. The other peak corresponded in mass to a Sec-to-Gln and/or Sec-to-Lys variant (54,649 Da, Fig.…”

Abiotic Stress Resistance, a Novel Moonlighting Function of Ribosomal Protein RPL44 in the Halophilic Fungus Aspergillus glaucus

“…However, recent studies have shown that some ribosomal proteins may also participate in cellular events apart from protein biosynthesis, and this has inspired research interest in the genes that encode them. Such extraribosomal functions include DNA reparation, RNA chaperone activity, cell development, regulation of differentiation, pathogen resistance mechanisms, and tumor repression (10)(11)(12)(13)(14). RPS3e is involved in DNA repair and selective gene regulation via the NF-B signaling pathway (15).…”

Translational Defects in a Mutant Deficient in YajL, the Bacterial Homolog of the Parkinsonism-Associated Protein DJ-1

“…L6 defects indirectly affect proofreading and result in decreased translation accuracy (14). L11 mutations result in defects in the binding of translation factors and in decreased translation accuracy (8), and L14 displays functional interactions with proteins L19 and S12 (the most important ribosomal protein for translation accuracy) (25).…”