scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 130-145 ◽  
Author(s):  
G. Blobel ◽  
D. D. Sabatini
Keyword(s):  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Subunit Structure ◽  
Amino Acid Residues ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Cell Fractions ◽  
Carboxy Terminal

Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals CONTROLLED PROTEOLYSIS OF NASCENT POLYPEPTIDES IN RAT LIVER CELL FRACTIONS

1970 ◽  
Vol 45 (1) ◽  
pp. 146-157 ◽  
Author(s):  
D. D. Sabatini ◽  
G. Blobel
Keyword(s):  
Amino Acid ◽  
Messenger Rna ◽  
Close Association ◽  
Membrane Integrity ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Cell Fractions

Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.

scholarly journals Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

2016 ◽  
Vol 113 (7) ◽  
pp. E829-E838 ◽  
Author(s):  
Yuhei Chadani ◽  
Tatsuya Niwa ◽  
Shinobu Chiba ◽  
Hideki Taguchi ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Ribosome Profiling ◽  
Cytosolic Proteins ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Chemical Trait

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study,Escherichia coliSecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members ofE.colithat were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

scholarly journals Comparative Analysis of Structural and Dynamical Features of Ribosome Upon Association With mRNA Reveals Potential Role of Ribosomal Proteins

2021 ◽  
Vol 8 ◽  
Author(s):  
Sneha Bheemireddy ◽  
Sankaran Sandhya ◽  
Narayanaswamy Srinivasan
Keyword(s):  
Protein Interactions ◽  
Messenger Rna ◽  
Ribosomal Proteins ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Network Models ◽  
Head Region ◽  
Initiation Factors ◽  
Dynamical Features ◽  
Mrna Binding

Ribosomes play a critical role in maintaining cellular proteostasis. The binding of messenger RNA (mRNA) to the ribosome regulates kinetics of protein synthesis. To generate an understanding of the structural, mechanistic, and dynamical features of mRNA recognition in the ribosome, we have analysed mRNA-protein interactions through a structural comparison of the ribosomal complex in the presence and absence of mRNA. To do so, we compared the 3-Dimensional (3D) structures of components of the two assembly structures and analysed their structural differences because of mRNA binding, using elastic network models and structural network-based analysis. We observe that the head region of 30S ribosomal subunit undergoes structural displacement and subunit rearrangement to accommodate incoming mRNA. We find that these changes are observed in proteins that lie far from the mRNA-protein interface, implying allostery. Further, through perturbation response scanning, we show that the proteins S13, S19, and S20 act as universal sensors that are sensitive to changes in the inter protein network, upon binding of 30S complex with mRNA and other initiation factors. Our study highlights the significance of mRNA binding in the ribosome complex and identifies putative allosteric sites corresponding to alterations in structure and/or dynamics, in regions away from mRNA binding sites in the complex. Overall, our work provides fresh insights into mRNA association with the ribosome, highlighting changes in the interactions and dynamics of the ribosome assembly because of the binding.

The carboxy-terminal region of mammalian HSP90 is required for its dimerization and function in vivo

1994 ◽  
Vol 14 (2) ◽  
pp. 1459-1464
Author(s):  
Y Minami ◽  
Y Kimura ◽  
H Kawasaki ◽  
K Suzuki ◽  
I Yahara
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Full Length ◽  
Yeast Cells ◽  
Amino Acid Residues ◽  
Haploid Strain ◽  
Carboxy Terminal Region ◽  
Carboxy Terminal ◽  
Hsp90 Alpha

The majority of mouse HSP90 exists as alpha-alpha and beta-beta homodimers. Truncation of the 15-kDa carboxy-terminal region of mouse HSP90 by digestion with the Ca(2+)-dependent protease m-calpain caused dissociation of the dimer. When expressed in a reticulocyte lysate, the full-length human HSP90 alpha formed a dimeric form. A plasmid harboring human HSP90 alpha cDNA was constructed so that the carboxy-terminal 49 amino acid residues were removed when translated in vitro. This carboxy-terminally truncated human HSP90 alpha was found to exist as a monomer. In contrast, loss of the 118 amino acid residues from the amino terminus of human HSP90 alpha did not affect its in vitro dimerization. Introduction of an expression plasmid harboring the full-length human HSP90 alpha complements the lethality caused by the double mutations of two HSP90-related genes, hsp82 and hsc82, in a haploid strain of Saccharomyces cerevisiae. The carboxy-terminally truncated human HSP90 alpha neither formed dimers in yeast cells nor rescued the lethal double mutant.

scholarly journals Eukaryotic Translation Initiation Factor 4AIII (eIF4AIII) Is Functionally Distinct from eIF4AI and eIF4AII

1999 ◽  
Vol 19 (11) ◽  
pp. 7336-7346 ◽  
Author(s):  
Qiyu Li ◽  
Hiroaki Imataka ◽  
Shigenobu Morino ◽  
George W. Rogers ◽  
Nancy J. Richter-Cook ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Rna Helicase ◽  
Amino Acid Identity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Eukaryotic Translation ◽  
Dependent Atpase ◽  
Eukaryotic Mrnas ◽  
Carboxy Terminal

ABSTRACT Eukaryotic initiation factor 4A (eIF4A) is an RNA-dependent ATPase and ATP-dependent RNA helicase that is thought to melt the 5′ proximal secondary structure of eukaryotic mRNAs to facilitate attachment of the 40S ribosomal subunit. eIF4A functions in a complex termed eIF4F with two other initiation factors (eIF4E and eIF4G). Two isoforms of eIF4A, eIF4AI and eIF4AII, which are encoded by two different genes, are functionally indistinguishable. A third member of the eIF4A family, eIF4AIII, whose human homolog exhibits 65% amino acid identity to human eIF4AI, has also been cloned from Xenopus and tobacco, but its function in translation has not been characterized. In this study, human eIF4AIII was characterized biochemically. While eIF4AIII, like eIF4AI, exhibits RNA-dependent ATPase activity and ATP-dependent RNA helicase activity, it fails to substitute for eIF4AI in an in vitro-reconstituted 40S ribosome binding assay. Instead, eIF4AIII inhibits translation in a reticulocyte lysate system. In addition, whereas eIF4AI binds independently to the middle and carboxy-terminal fragments of eIF4G, eIF4AIII binds to the middle fragment only. These functional differences between eIF4AI and eIF4AIII suggest that eIF4AIII might play an inhibitory role in translation under physiological conditions.

scholarly journals The folding and unfolding behavior of ribonuclease H on the ribosome

2020 ◽  
Vol 295 (33) ◽  
pp. 11410-11417 ◽  
Author(s):  
Madeleine K. Jensen ◽  
Avi J. Samelson ◽  
Annette Steward ◽  
Jane Clarke ◽  
Susan Marqusee
Keyword(s):  
Protein Stability ◽  
Ribosomal Proteins ◽  
Profile Analysis ◽  
Rnase H ◽  
Folding Intermediate ◽  
Folding Rate ◽  
Nascent Chain ◽  
Force Profile ◽  
Pulse Proteolysis

The health of a cell depends on accurate translation and proper protein folding, whereas misfolding can lead to aggregation and disease. The first opportunity for a protein to fold occurs during translation, when the ribosome and surrounding environment can affect the nascent chain energy landscape. However, quantifying these environmental effects is challenging because ribosomal proteins and rRNA preclude most spectroscopic measurements of protein energetics. Here, we have applied two gel-based approaches, pulse proteolysis and force-profile analysis, to probe the folding and unfolding pathways of RNase H (RNH) nascent chains stalled on the prokaryotic ribosome in vitro. We found that ribosome-stalled RNH has an increased unfolding rate compared with free RNH. Because protein stability is related to the ratio of the unfolding and folding rates, this increase completely accounts for the observed change in protein stability and indicates that the folding rate is unchanged. Using arrest peptide–based force-profile analysis, we assayed the force generated during the folding of RNH on the ribosome. Surprisingly, we found that population of the RNH folding intermediate is required to generate sufficient force to release a stall induced by the SecM stalling sequence and that readthrough of SecM directly correlates with the stability of the RNH folding intermediate. Together, these results imply that the folding pathway of RNH is unchanged on the ribosome. Furthermore, our findings indicate that the ribosome promotes RNH unfolding while the nascent chain is proximal to the ribosome, which may limit the deleterious effects of RNH misfolding and assist in folding fidelity.

scholarly journals Basic amino acid residue cluster within nuclear targeting sequence motif is essential for cytoplasmic plectin-vimentin network junctions.

1996 ◽  
Vol 134 (6) ◽  
pp. 1455-1467 ◽  
Author(s):  
B Nikolic ◽  
E Mac Nulty ◽  
B Mir ◽  
G Wiche
Keyword(s):  
Amino Acid ◽  
Intermediate Filaments ◽  
Basic Amino Acid ◽  
Site Directed Mutagenesis ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Nuclear Targeting ◽  
Mutant Proteins ◽  
Carboxy Terminal

We have generated a series of plectin deletion and mutagenized cDNA constructs to dissect the functional sequences that mediate plectin's interaction with intermediate filament (IF) networks, and scored their ability to coalign or disrupt intermediate filaments when ectopically expressed in rat kangaroo PtK2 cells. We show that a stretch of approximately 50 amino acid residues within plectin's carboxy-terminal repeat 5 domain serves as a unique binding site for both vimentin and cytokeratin IF networks of PtK2 cells. Part of the IF-binding domain was found to constitute a functional nuclear localization signal (NLS) motif, as demonstrated by nuclear import of cytoplasmic proteins linked to this sequence. Site directed mutagenesis revealed a specific cluster of four basic amino acid residues (arg4277-arg4280) residing within the NLS sequence motif to be essential for IF binding. When mutant proteins corresponding to those expressed in PtK2 cells were expressed in bacteria and purified proteins subjected to a sensitive quantitative overlay binding assay using Eu3+-labeled vimentin, the relative binding capacities of mutant proteins measured were fully consistent with the mutant's phenotypes observed in living cells. Using recombinant proteins we also show by negative staining and rotary shadowing electron microscopy that in vitro assembled vimentin intermediate filaments become packed into dense aggregates upon incubation with plectin repeat 5 domain, in contrast to repeat 4 domain or a mutated repeat 5 domain.

scholarly journals Nascent Polypeptide–associated Complex Stimulates Protein Import into Yeast Mitochondria

1999 ◽  
Vol 10 (10) ◽  
pp. 3289-3299 ◽  
Author(s):  
Ursula Fünfschilling ◽  
Sabine Rospert
Keyword(s):  
Protein Import ◽  
Surface Proteins ◽  
Yeast Mitochondria ◽  
Nascent Polypeptide ◽  
C Terminus ◽  
Precursor Proteins ◽  
Nascent Chain ◽  
Stimulatory Factor ◽  
Import System

To identify yeast cytosolic proteins that mediate targeting of precursor proteins to mitochondria, we developed an in vitro import system consisting of purified yeast mitochondria and a radiolabeled mitochondrial precursor protein whose C terminus was still attached to the ribosome. In this system, the N terminus of the nascent chain was translocated across both mitochondrial membranes, generating a translocation intermediate spanning both membranes. The nascent chain could then be completely chased into the mitochondrial matrix after release from the ribosome. Generation of this import intermediate was dependent on a mitochondrial membrane potential, mitochondrial surface proteins, and was stimulated by proteins that could be released from the ribosomes by high salt. The major salt-released stimulatory factor was yeast nascent polypeptide–associated complex (NAC). Purified NAC fully restored import of salt-washed ribosome-bound nascent chains by enhancing productive binding of the chains to mitochondria. We propose that ribosome-associated NAC facilitates recognition of nascent precursor chains by the mitochondrial import machinery.

scholarly journals cfrB, cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

2019 ◽  
Author(s):  
Vanja Stojković ◽  
María Fernanda Ulate ◽  
Fanny Hidalgo-Villeda ◽  
Emmanuel Aguilar ◽  
Camilo Monge-Cascante ◽  
...  
Keyword(s):  
Latin America ◽  
Ribosomal Proteins ◽  
Functional Characterization ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Cross Resistance ◽  
Protection Factor ◽  
E Coli

ABSTRACTCfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

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