scholarly journals An intermediate state of bacterial ribosome dissociation is fixed by yeast mitochondrial initiation factor 3

2018 ◽  
Author(s):  
Sergey Levitskii ◽  
Ksenia Derbikova ◽  
Andrey V Golovin ◽  
Anton Kuzmenko ◽  
Maria V Baleva ◽  
...  
Keyword(s):  
Intermediate State ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Sedimentation Coefficient ◽  
Initiation Factor ◽  
Bacterial Cells ◽  
Ribosomal Subunits ◽  
Bacterial Ribosome ◽  
Dissociated State

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.

scholarly journals An intermediate state of bacterial ribosome dissociation is fixed by yeast mitochondrial initiation factor 3

2018 ◽  
Author(s):  
Sergey Levitskii ◽  
Ksenia Derbikova ◽  
Andrey V Golovin ◽  
Anton Kuzmenko ◽  
Maria V Baleva ◽  
...  
Keyword(s):  
Intermediate State ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Sedimentation Coefficient ◽  
Initiation Factor ◽  
Bacterial Cells ◽  
Ribosomal Subunits ◽  
Bacterial Ribosome ◽  
Dissociated State

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with the large subunits. In this work, we have exchanged IF3 in E.coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E.coli ribosomes into subunits. Instead, it fixes an intermediate state of ribosomes dissociation characterized by sedimentation coefficient about 60S. Using molecular modeling, we show that such fixation is due to mitochondria-specific terminal extensions of Aim23p that stabilize the position of the protein on the bacterial ribosome.

scholarly journals 60S dynamic state of bacterial ribosome is fixed by yeast mitochondrial initiation factor 3

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5620 ◽  
Author(s):  
Sergey Levitskii ◽  
Ksenia Derbikova ◽  
Maria V. Baleva ◽  
Anton Kuzmenko ◽  
Andrey V. Golovin ◽  
...  
Keyword(s):  
Dynamic Equilibrium ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Sedimentation Coefficient ◽  
Initiation Factor ◽  
Dynamic State ◽  
Bacterial Cells ◽  
E Coli ◽  
Bacterial Ribosome

The processes of association and dissociation of ribosomal subunits are of great importance for the protein biosynthesis. The mechanistic details of these processes, however, are not well known. In bacteria, upon translation termination, the ribosome dissociates into subunits which is necessary for its further involvement into new initiation step. The dissociated state of the ribosome is maintained by initiation factor 3 (IF3) which binds to free small subunits and prevents their premature association with large subunits. In this work, we have exchanged IF3 in Escherichia coli cells by its ortholog from Saccharomyces cerevisiae mitochondria (Aim23p) and showed that yeast protein cannot functionally substitute the bacterial one and is even slightly toxic for bacterial cells. Our in vitro experiments have demonstrated that Aim23p does not split E. coli ribosomes into subunits. Instead, it fixes a state of ribosomes characterized by sedimentation coefficient about 60S which is not a stable structure but rather reflects a shift of dynamic equilibrium between associated and dissociated states of the ribosome. Mitochondria-specific terminal extensions of Aim23p are necessary for “60S state” formation, and molecular modeling results point out that these extensions might stabilize the position of the protein on the bacterial ribosome.

scholarly journals CTELS: A Cell-Free System for the Analysis of Translation Termination Rate

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 911 ◽  
Author(s):  
Kseniya A. Lashkevich ◽  
Valeriya I. Shlyk ◽  
Artem S. Kushchenko ◽  
Vadim N. Gladyshev ◽  
Elena Z. Alkalaeva ◽  
...  
Keyword(s):  
Stop Codon ◽  
Translation Termination ◽  
Protein Biosynthesis ◽  
Inhibitory Effect ◽  
In Vitro Translation ◽  
Nonsense Suppression ◽  
Translation System ◽  
Free System ◽  
Termination Rate

Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3′ UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a “leaky” stop codon context, which likely defines the basis of nonsense suppression.

scholarly journals Lack of inhibition by l-1-chloro-4-phenyl-3-toluene-p-sulphoamidobutan-2-one (l-1-tosylamido-2-phenylethyl chloromethyl ketone) of the formation of bacterial polypeptide chain initiation complex (Short Communication)

1973 ◽  
Vol 136 (2) ◽  
pp. 433-436 ◽  
Author(s):  
J. Jonák ◽  
B. F. C. Clark
Keyword(s):  
Short Communication ◽  
Polypeptide Chain ◽  
Protein Biosynthesis ◽  
Elongation Factor ◽  
Initiation Factor ◽  
Chain Elongation ◽  
Initiation Complex ◽  
Chloromethyl Ketone ◽  
Chain Initiation

The chymotrypsin inhibitor l-1-chloro-4-phenyl-3-toluene-p-sulphonamidobutan-2-one does not inhibit the function of the initiation factor during the formation of the polypeptide chain initiation complex in vitro. Since the inhibitor has been shown previously to inhibit polypeptide chain elongation by reacting with elongation factor EF-Tu, the inhibitor can be used to investigate the initiation and elongation steps of protein biosynthesis separately.

Expression of antisense RNA against initiation factor eIF-4E mRNA in HeLa cells results in lengthened cell division times, diminished translation rates, and reduced levels of both eIF-4E and the p220 component of eIF-4F

1991 ◽  
Vol 11 (11) ◽  
pp. 5435-5445
Author(s):  
A De Benedetti ◽  
S Joshi-Barve ◽  
C Rinker-Schaeffer ◽  
R E Rhoads
Keyword(s):  
Hela Cells ◽  
Antisense Rna ◽  
Inducible Promoter ◽  
Initiation Factor ◽  
Rna Expression ◽  
Concomitant Increase ◽  
Ribosomal Subunits ◽  
Protein Levels

HeLa cells were transformed to express antisense RNA against initiation factor eIF-4E mRNA from an inducible promoter. In the absence of inducer, these cells (AS cells) were morphologically similar to control cells but grew four- to sevenfold more slowly. Induction of antisense RNA production was lethal. Both eIF-4E mRNA and protein levels were reduced in proportion to the degree of antisense RNA expression, as were the rates of protein synthesis in vivo and in vitro. Polysomes were disaggregated with a concomitant increase in ribosomal subunits. Translation in vitro was restored by addition of the initiation factor complex eIF-4F but not by eIF-4E alone. Immunological analysis revealed that the p220 component of eIF-4F was decreased in extracts of AS cells and undetectable in AS cells treated with inducer, suggesting that p220 and eIF-4E levels are coordinately regulated. eIF-4A, another component of eIF-4F, was unaltered.

Effect of starvation on initiation of protein synthesis in skeletal muscle and heart.

1978 ◽  
Vol 235 (2) ◽  
pp. E126 ◽  
Author(s):  
D E Rannels ◽  
A E Pegg ◽  
S R Rannels ◽  
L S Jefferson
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Heart Muscle ◽  
Psoas Muscle ◽  
Peptide Chain ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Chain Initiation ◽  
Rate Limiting

Psoas muscle of rats starved for 2 or 4 days contained increased levels of ribosomal subunits and exhibited reduced rates of protein synthesis in vitro, demonstrating a starvation-induced inhibition of peptide-chain initiation. The activity of an eIF-2-like initiation factor, assayed in postribosomal supernatants, decreased in psoas during starvation, parallel to a 25% reduction in the RNA level. Reduced eIF-2 activity did not result from nucleotide depletion or increased deacylation of initiator tRNA, nor was it abolished by extensive dialysis. Perfusion of psoas muscle in the presence of insulin reversed the starvation-induced block in peptide-chain initiation, but did not alter the activity of eIF-2 or level of RNA. Furthermore, heart muscle did not manifest a starvation-induced block in peptide-chain initiation even though the activity of eIF-2 and the level of RNA decreased as a result of food deprivation. Thus loss of eIF 2 activity in psoas and heart did not parallel changes in peptide-chain initiation but was associated with a reduction in tissue RNA. These results indicate that the level of eIF-2 is not rate-limiting for peptide-chain initiation under the conditions tested in this study.

scholarly journals The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

1999 ◽  
Vol 19 (2) ◽  
pp. 1416-1426 ◽  
Author(s):  
Kausik Si ◽  
Umadas Maitra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
Translation Initiation Factor ◽  
Yeast Cells ◽  
Initiation Factor ◽  
Ribosomal Subunits

ABSTRACT Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M r 25,550), designated TIF6, has been cloned and expressed inEscherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.

scholarly journals Expression of antisense RNA against initiation factor eIF-4E mRNA in HeLa cells results in lengthened cell division times, diminished translation rates, and reduced levels of both eIF-4E and the p220 component of eIF-4F.

1991 ◽  
Vol 11 (11) ◽  
pp. 5435-5445 ◽  
Author(s):  
A De Benedetti ◽  
S Joshi-Barve ◽  
C Rinker-Schaeffer ◽  
R E Rhoads
Keyword(s):  
Hela Cells ◽  
Antisense Rna ◽  
Inducible Promoter ◽  
Initiation Factor ◽  
Rna Expression ◽  
Concomitant Increase ◽  
Ribosomal Subunits ◽  
Protein Levels

HeLa cells were transformed to express antisense RNA against initiation factor eIF-4E mRNA from an inducible promoter. In the absence of inducer, these cells (AS cells) were morphologically similar to control cells but grew four- to sevenfold more slowly. Induction of antisense RNA production was lethal. Both eIF-4E mRNA and protein levels were reduced in proportion to the degree of antisense RNA expression, as were the rates of protein synthesis in vivo and in vitro. Polysomes were disaggregated with a concomitant increase in ribosomal subunits. Translation in vitro was restored by addition of the initiation factor complex eIF-4F but not by eIF-4E alone. Immunological analysis revealed that the p220 component of eIF-4F was decreased in extracts of AS cells and undetectable in AS cells treated with inducer, suggesting that p220 and eIF-4E levels are coordinately regulated. eIF-4A, another component of eIF-4F, was unaltered.

scholarly journals Mode of Action of Ranbezolid against Staphylococci and Structural Modeling Studies of Its Interaction with Ribosomes

2008 ◽  
Vol 53 (4) ◽  
pp. 1427-1433 ◽  
Author(s):  
Vandana Kalia ◽  
Rajni Miglani ◽  
Kedar P. Purnapatre ◽  
Tarun Mathur ◽  
Smita Singhal ◽  
...  
Keyword(s):  
Wide Spectrum ◽  
Protein Biosynthesis ◽  
Membrane Integrity ◽  
Lipid Synthesis ◽  
In Vitro Translation ◽  
Gram Positive Bacteria ◽  
Modeling Studies ◽  
Bacterial Ribosome ◽  
Bacterial Protein Synthesis

ABSTRACT Oxazolidinones are known to inhibit protein biosynthesis and act against a wide spectrum of gram-positive bacteria. A new investigational oxazolidinone, ranbezolid, inhibited bacterial protein synthesis in Staphylococcus aureus and Staphylococcus epidermidis. In S. epidermidis, ranbezolid showed inhibition of cell wall and lipid synthesis and a dose-dependent effect on membrane integrity. A kill-kinetics study showed that ranbezolid was bactericidal against S. epidermidis. In vitro translation of the luciferase gene done using bacterial and mammalian ribosomes indicated that ranbezolid specifically inhibited the bacterial ribosome. Molecular modeling studies revealed that both linezolid and ranbezolid fit in similar manners the active site of ribosomes, with total scores, i.e., theoretical binding affinities after consensus, of 5.2 and 6.9, respectively. The nitrofuran ring in ranbezolid is extended toward C2507, G2583, and U2584, and the nitro group forms a hydrogen bond from the base of G2583. The interaction of ranbezolid with the bacterial ribosomes clearly helps to elucidate its potent activity against the target pathogen.

scholarly journals Discovery of a small molecule that inhibits bacterial ribosome biogenesis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jonathan M Stokes ◽  
Joseph H Davis ◽  
Chand S Mangat ◽  
James R Williamson ◽  
Eric D Brown
Keyword(s):  
Small Molecule ◽  
Ribosome Biogenesis ◽  
Anticonvulsant Drug ◽  
Protein Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Bacterial Ribosome ◽  
Cold Sensitive

While small molecule inhibitors of the bacterial ribosome have been instrumental in understanding protein translation, no such probes exist to study ribosome biogenesis. We screened a diverse chemical collection that included previously approved drugs for compounds that induced cold sensitive growth inhibition in the model bacterium Escherichia coli. Among the most cold sensitive was lamotrigine, an anticonvulsant drug. Lamotrigine treatment resulted in the rapid accumulation of immature 30S and 50S ribosomal subunits at 15°C. Importantly, this was not the result of translation inhibition, as lamotrigine was incapable of perturbing protein synthesis in vivo or in vitro. Spontaneous suppressor mutations blocking lamotrigine activity mapped solely to the poorly characterized domain II of translation initiation factor IF2 and prevented the binding of lamotrigine to IF2 in vitro. This work establishes lamotrigine as a widely available chemical probe of bacterial ribosome biogenesis and suggests a role for E. coli IF2 in ribosome assembly.

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