Genetic probes of ribosomal RNA function

1995 ◽  
Vol 73 (11-12) ◽  
pp. 859-868 ◽  
Author(s):  
Michael O'Connor ◽  
Carleen A. Brunelli ◽  
Matthew A. Firpo ◽  
Steven T. Gregory ◽  
Kathy R. Lieberman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Rna ◽  
Phylogenetic Analyses ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Genetic Approach ◽  
Functional Roles ◽  
Genetic Probes ◽  
4.5S Rna

We have used a genetic approach to uncover the functional roles of rRNA in protein synthesis. Mutations were constructed in a cloned rrn operon by site-directed mutagenesis or isolated by genetic selections following random mutagenesis. We have identified mutations that affect each step in the process of translation. The data are consistent with the results of biochemical and phylogenetic analyses but, in addition, have provided novel information on regions of rRNA not previously investigated.Key words: decoding, peptidyltransferase, streptomycin, paromomycin, suppression, 4.5S RNA.

ΔLys120, a mutation which destabilizes the ribosome-binding domain of ribosomal protein L7/L12

1990 ◽  
Vol 68 (5) ◽  
pp. 832-838 ◽  
Author(s):  
Michael Laughrea ◽  
Eric Higgins
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Ribosome Binding ◽  
Mutant Genes

Five-residue-long deletions centered on A1a63, A1a75, and Glu118 of ribosomal protein L7/L12 gave low mutant yields (5% or less) when the mutant genes were cloned in phage M13mp18 and controlled by the L10 promotor. Deletions of Glu118–Lys120 or Lys120 (the COOH-terminus of L7/L12) gave higher mutant yields, up to 50% with L7/L12ΔLys120. L7/L12ΔLys120 was not preferentially found in the S100 and not preferentially removed by LiCl washing, but was preferentially extracted from 70S ribosomes in the presence of 28–35% ethanol in 0.25–0.5 M NH4Cl. It follows that ΔLys120 destabilizes the ribosome-binding domain of ribosomal protein L7/L12 in an ethanol-containing solvent, which raises the question whether Lys120 is part of the ribosome-binding domain of L7/L12 during some step of protein synthesis or whether it is essential to preserve the conformation of the physiological ribosome-binding domain under structurally stressful conditions.Key words: ribosome, ribosomal protein L7/L12, site-directed mutagenesis.

scholarly journals Schistosome-derived omega-1 drives Th2 polarization by suppressing protein synthesis following internalization by the mannose receptor

2012 ◽  
Vol 209 (10) ◽  
pp. 1753-1767 ◽  
Author(s):  
Bart Everts ◽  
Leonie Hussaarts ◽  
Nicole N. Driessen ◽  
Moniek H.J. Meevissen ◽  
Gabriele Schramm ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Protein Synthesis ◽  
Messenger Rna ◽  
Molecular Mechanisms ◽  
Mannose Receptor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Th2 Polarization ◽  
Th2 Responses ◽  
Egg Antigen

Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

scholarly journals Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

2016 ◽  
Vol 473 (11) ◽  
pp. 1605-1615 ◽  
Author(s):  
Sara R. Costa ◽  
Magdalena Marek ◽  
Kristian B. Axelsen ◽  
Lisa Theorin ◽  
Thomas G. Pomorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Post Translational Modifications ◽  
Functional Roles ◽  
Evolutionarily Conserved ◽  
A Site ◽  
P Type ◽  
Cell Division Control

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn181 and Asn231. Whereas mutation of Asn231 seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn181 disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys86 and Cys107 compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys158 and Cys172 has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase.

Site-directed mutagenesis of ribosomal RNA

1982 ◽  
Vol 159 (3) ◽  
pp. 417-439 ◽  
Author(s):  
Michael J.R. Stark ◽  
Richard L. Gourse ◽  
Albert E. Dahlberg
Keyword(s):  
Ribosomal Rna ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

1, 10/1, 11 Cyclization catalyzed by diverged plant sesquiterpene synthases is dependent on a single residue

2021 ◽  
Author(s):  
Xin Fang ◽  
Jin-Quan Huang ◽  
Dong-Mei Li ◽  
Jian-Xu Li ◽  
Jia-Ling Lin ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Site Directed Mutagenesis ◽  
Terpene Synthase ◽  
Directed Mutagenesis

The exquisite chemodiversity of terpenoids are the product of the large diverse terpene synthase (TPS) superfamily. Here, by using structural and phylogenetic analyses and site-directed mutagenesis, we identified a residue...

scholarly journals Cumulative Effect of Amino Acid Replacements Results in Enhanced Thermostability of Potato Type L α-Glucan Phosphorylase

2005 ◽  
Vol 71 (9) ◽  
pp. 5433-5439 ◽  
Author(s):  
Michiyo Yanase ◽  
Hiroki Takata ◽  
Kazutoshi Fujii ◽  
Takeshi Takaha ◽  
Takashi Kuriki
Keyword(s):  
Heat Treatment ◽  
Random Mutagenesis ◽  
Cumulative Effect ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Triple Mutant ◽  
Original Activity ◽  
Glucan Phosphorylase

ABSTRACT The thermostability of potato type L α-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations—Phe39→Leu (F39L), Asn135→Ser (N135S), and Thr706→Ile (T706I)—by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60°C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65°C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.

Sign in / Sign up

Export Citation Format

Share Document