scholarly journals Functional implications of ribosomal protein L2 in protein biosynthesis as shown by in vivo replacement studies

1998 ◽  
Vol 331 (2) ◽  
pp. 423-430 ◽  
Author(s):  
Monika ÜHLEIN ◽  
Wolfgang WEGLÖHNER ◽  
Henning URLAUB ◽  
Brigitte WITTMANN-LIEBOLD
Keyword(s):  
Ribosomal Proteins ◽  
Protein Biosynthesis ◽  
Complex Structure ◽  
Peptide Bond ◽  
Histidine Residue ◽  
Peptide Bond Formation ◽  
Homologous Proteins ◽  
Translational Activity ◽  
Translational Apparatus

The translational apparatus is a highly complex structure containing three to four RNA molecules and more than 50 different proteins. In recent years considerable evidence has accumulated to indicate that the RNA participates intensively in the catalysis of peptide-bond formation, whereas a direct involvement of the ribosomal proteins has yet to be demonstrated. Here we report the functional and structural conservation of a peptidyltransferase centre protein in all three phylogenetic domains. In vivo replacement studies show that the Escherichia coli L2 protein can be replaced by its homologous proteins from human and archaebacterial ribosomes. These hybrid ribosomes are active in protein biosynthesis, as proven by polysome analysis and poly(U)-dependent polyphenylalanine synthesis. Furthermore, we demonstrate that a specific, highly conserved, histidine residue in the C-terminal region of L2 is essential for the function of the translational apparatus. Replacement of this histidine residue in the human and archaebacterial proteins by glycine, arginine or alanine had no effect on ribosome assembly, but strongly reduced the translational activity of ribosomes containing these mutants.

scholarly journals Ser/Thr phospho-regulation by PknB and Stp mediates bacterial quiescence and antibiotic persistence in Staphylococcus aureus

2021 ◽  
Author(s):  
Markus Huemer ◽  
Srikanth Mairpady Shambat ◽  
Sandro Pereira ◽  
Lies Van Gestel ◽  
Judith Bergada-Pijuan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ribosomal Proteins ◽  
Environmental Changes ◽  
Acid Stress ◽  
Elongation Factor ◽  
Growth Delay ◽  
Lag Phase ◽  
Cellular Processes ◽  
Translational Activity

Staphylococcus aureus colonizes 30 to 50% of healthy adults and can cause a variety of diseases, ranging from superficial to life-threatening invasive infections such as bacteraemia and endocarditis. Often, these infections are chronic and difficult-to-treat despite adequate antibiotic therapy. Most antibiotics act on metabolically active bacteria in order to eradicate them. Thus, bacteria with minimized energy consumption resulting in metabolic quiescence, have increased tolerance to antibiotics. The most energy intensive process in cells - protein synthesis - is attenuated in bacteria entering into quiescence. Eukaryote-like serine/threonine kinases (STKs) and phosphatases (STPs) can fine-tune essential cellular processes, thereby enabling bacteria to quickly respond to environmental changes and to modulate quiescence. Here, we show that deletion of the only annotated functional STP, named Stp, in S. aureus leads to increased bacterial lag-phase and phenotypic heterogeneity under different stress challenges, including acidic pH, intracellular milieu and in vivo abscess environment. This growth delay was associated with reduced intracellular ATP levels and increased antibiotic persistence. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified possible targets of Ser/Thr phosphorylation that regulate cellular processes and bacterial growth, such as ribosomal proteins including the essential translation elongation factor EF-G. Finally, we show that acid stress leads to a reduced translational activity in the stp deletion mutant indicating metabolic quiescence correlating with increased antibiotic persistence.

scholarly journals Crystal structure of eukaryotic ribosome and its complexes with inhibitors

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160184 ◽  
Author(s):  
Gulnara Yusupova ◽  
Marat Yusupov
Keyword(s):  
Crystal Structure ◽  
Ribosomal Rna ◽  
Protein Biosynthesis ◽  
Protein Translation ◽  
Peptide Bond ◽  
Binding Mode ◽  
High Eukaryote ◽  
Peptide Bond Formation ◽  
80S Ribosome ◽  
Eukaryotic Ribosome

A high-resolution structure of the eukaryotic ribosome has been determined and has led to increased interest in studying protein biosynthesis and regulation of biosynthesis in cells. The functional complexes of the ribosome crystals obtained from bacteria and yeast have permitted researchers to identify the precise residue positions in different states of ribosome function. This knowledge, together with electron microscopy studies, enhances our understanding of how basic ribosome processes, including mRNA decoding, peptide bond formation, mRNA, and tRNA translocation and cotranslational transport of the nascent peptide, are regulated. In this review, we discuss the crystal structure of the entire 80S ribosome from yeast, which reveals its eukaryotic-specific features, and application of X-ray crystallography of the 80S ribosome for investigation of the binding mode for distinct compounds known to inhibit or modulate the protein-translation function of the ribosome. We also refer to a challenging aspect of the structural study of ribosomes, from higher eukaryotes, where the structures of major distinctive features of higher eukaryote ribosome—the high-eukaryote–specific long ribosomal RNA segments (about 1MDa)—remain unresolved. Presently, the structures of the major part of these high-eukaryotic expansion ribosomal RNA segments still remain unresolved. This article is part of the themed issue ‘Perspectives on the ribosome’.

Purification of factor EF-P, a protein that stimulates peptide-bond synthesis with certain aminoacyl-tRNA analogues

1979 ◽  
Vol 57 (6) ◽  
pp. 749-757 ◽  
Author(s):  
Bernard R. Glick ◽  
Robert M. Green ◽  
M. Clelia Ganoza
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein Biosynthesis ◽  
Bond Formation ◽  
Frictional Coefficient ◽  
Peptide Bond ◽  
Asymmetric Structure ◽  
Peptide Bond Formation ◽  
Apparent Homogeneity ◽  
Stokes Radius

Factor EF-P is a nonribosomal (soluble) protein of Escherichia coli that stimulates peptide bond synthesis when certain aminoacyl-tRNA analogues are used. The purification of this protein to apparent homogeneity is described here. EF-P has a molecular weight of about 21 000, a Stokes radius of 27 Å (1 Å = 0.1 nm), and a frictional coefficient of 1.48, suggesting an asymmetric structure. By this and a number of other criteria, EF-P is a new factor that controls peptide bond formation during protein biosynthesis.

scholarly journals Yeast Asc1p and Mammalian RACK1 Are Functionally Orthologous Core 40S Ribosomal Proteins That Repress Gene Expression

2004 ◽  
Vol 24 (18) ◽  
pp. 8276-8287 ◽  
Author(s):  
Vincent R. Gerbasi ◽  
Connie M. Weaver ◽  
Salisha Hill ◽  
David B. Friedman ◽  
Andrew J. Link
Keyword(s):  
Gene Expression ◽  
Translational Control ◽  
Ribosomal Proteins ◽  
Translational Activity ◽  
Eukaryotic Gene ◽  
Associated Proteins ◽  
Yeast Ribosomes ◽  
Specific Proteins ◽  
Conserved Core

ABSTRACT Translation of mRNA into protein is a fundamental step in eukaryotic gene expression requiring the large (60S) and small (40S) ribosome subunits and associated proteins. By modern proteomic approaches, we previously identified a novel 40S-associated protein named Asc1p in budding yeast and RACK1 in mammals. The goals of this study were to establish Asc1p or RACK1 as a core conserved eukaryotic ribosomal protein and to determine the role of Asc1p or RACK1 in translational control. We provide biochemical, evolutionary, genetic, and functional evidence showing that Asc1p or RACK1 is indeed a conserved core component of the eukaryotic ribosome. We also show that purified Asc1p-deficient ribosomes have increased translational activity compared to that of wild-type yeast ribosomes. Further, we demonstrate that asc1Δ null strains have increased levels of specific proteins in vivo and that this molecular phenotype is complemented by either Asc1p or RACK1. Our data suggest that one of Asc1p's or RACK1's functions is to repress gene expression.

scholarly journals Rχ-01, a New Family of Oxazolidinones That Overcome Ribosome-Based Linezolid Resistance

2008 ◽  
Vol 52 (10) ◽  
pp. 3550-3557 ◽  
Author(s):  
Eugene Skripkin ◽  
Timothy S. McConnell ◽  
Joseph DeVito ◽  
Laura Lawrence ◽  
Joseph A. Ippolito ◽  
...  
Keyword(s):  
Peptide Bond ◽  
Nonsense Suppression ◽  
Intrinsic Activity ◽  
Resistant Bacteria ◽  
Peptide Bond Formation ◽  
New Family ◽  
Linezolid Resistance ◽  
A Site

ABSTRACT New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrug-resistant bacteria. In this study, we characterized several members of a new oxazolidinone family, Rχ-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the Rχ-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether Rχ-01 compounds protect U2585 (Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The Rχ-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the Rχ-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the Rχ-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the Rχ-01 family of compounds is currently undergoing clinical trials.

scholarly journals Alkylative damage of mRNA leads to ribosome stalling and rescue by trans translation in bacteria

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Erica N Thomas ◽  
Kyusik Q Kim ◽  
Emily P McHugh ◽  
Thomas Marcinkiewicz ◽  
Hani S Zaher
Keyword(s):  
Messenger Rna ◽  
Alkylating Agents ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
In Vitro System ◽  
Ribosome Stalling ◽  
Small Chemical ◽  
Chemical Integrity

Similar to DNA replication, translation of the genetic code by the ribosome is hypothesized to be exceptionally sensitive to small chemical changes to its template mRNA. Here we show that the addition of common alkylating agents to growing cultures of Escherichia coli leads to the accumulation of several adducts within RNA, including N(1)-methyladenosine (m1A). As expected, the introduction of m1A to model mRNAs was found to reduce the rate of peptide bond formation by three orders of magnitude in a well-defined in vitro system. These observations suggest that alkylative stress is likely to stall translation in vivo and necessitates the activation of ribosome-rescue pathways. Indeed, the addition of alkylation agents was found to robustly activate the transfer-messenger RNA system, even when transcription was inhibited. Our findings suggest that bacteria carefully monitor the chemical integrity of their mRNA and they evolved rescue pathways to cope with its effect on translation.

Peptidyl transferase and beyond

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1041-1047 ◽  
Author(s):  
Jacek Wower ◽  
Iwona K. Wower ◽  
Stanislav V. Kirillov ◽  
Kirill V. Rosen ◽  
Robert A. Zimmermann ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Peptide Bond ◽  
23S Rrna ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
The Past ◽  
And Function ◽  
Trna Binding

The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3′ end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.Key words: peptidyl transferase, ribosome, tRNA, photoreactive nucleos/tides, crosslinking.

scholarly journals Structure and functions of 5S rRNA.

2001 ◽  
Vol 48 (1) ◽  
pp. 191-198 ◽  
Author(s):  
M Z Barciszewska ◽  
M Szymański ◽  
V A Erdmann ◽  
J Barciszewski
Keyword(s):  
Messenger Rna ◽  
Protein Biosynthesis ◽  
Large Subunit ◽  
Genetic Material ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Small Subunit ◽  
5S Rrna ◽  
23S Rrna ◽  
Peptide Bond Formation

The ribosome is a macromolecular assembly that is responsible for protein biosynthesis in all organisms. It is composed of two-subunit, ribonucleoprotein particles that translate the genetic material into an encoded polypeptides. The small subunit is the site of codon-anticodon interaction between the messenger RNA (mRNA) and transfer RNA (tRNA) substrates, and the large subunit catalyses peptide bond formation. The peptidyltransferase activity is fulfilled by 23S rRNA, which means that ribosome is a ribozyme. 5S rRNA is a conserved component of the large ribosomal subunit that is thought to enhance protein synthesis by stabilizing ribosome structure. This paper shortly summarises new results obtained on the structure and function of 5S rRNA.

scholarly journals Avilamycin and evernimicin induce structural changes in rProteins uL16 and CTC that enhance the inhibition of A-site tRNA binding

2016 ◽  
Vol 113 (44) ◽  
pp. E6796-E6805 ◽  
Author(s):  
Miri Krupkin ◽  
Itai Wekselman ◽  
Donna Matzov ◽  
Zohar Eyal ◽  
Yael Diskin Posner ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Protein Biosynthesis ◽  
Protein A ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Gram Positive ◽  
Gram Positive Bacteria

Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA—namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16—these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards.

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