Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 187
Filtrar
1.
Viruses ; 16(6)2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38932225

RESUMO

The innate immune system, particularly the interferon (IFN) system, constitutes the initial line of defense against viral infections. IFN signaling induces the expression of interferon-stimulated genes (ISGs), and their products frequently restrict viral infection. Retroviruses like the human immunodeficiency viruses and the human T-lymphotropic viruses cause severe human diseases and are targeted by ISG-encoded proteins. Here, we discuss ISGs that inhibit the translation of retroviral mRNAs and thereby retrovirus propagation. The Schlafen proteins degrade cellular tRNAs and rRNAs needed for translation. Zinc Finger Antiviral Protein and RNA-activated protein kinase inhibit translation initiation factors, and Shiftless suppresses translation recoding essential for the expression of retroviral enzymes. We outline common mechanisms that underlie the antiviral activity of multifunctional ISGs and discuss potential antiretroviral therapeutic approaches based on the mode of action of these ISGs.


Assuntos
Interferons , Biossíntese de Proteínas , Retroviridae , Humanos , Interferons/imunologia , Interferons/metabolismo , Interferons/genética , Retroviridae/genética , Retroviridae/fisiologia , Imunidade Inata , Animais , Transdução de Sinais , Infecções por Retroviridae/virologia , Infecções por Retroviridae/imunologia , Infecções por Retroviridae/genética
2.
Nucleic Acids Res ; 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38842944

RESUMO

The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.

3.
Nucleic Acids Res ; 52(11): 6507-6517, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38783118

RESUMO

The ribosome can slide along mRNA without establishing codon-anticodon interactions. This movement can be regulated (programmed) by the elements encoded in the mRNA, as observed in bypassing of non-coding gap in gene 60 of bacteriophage T4, or occur spontaneously, such as during traversal by the 70S ribosome of the 3'UTRs or upon re-initiation on bacterial polycistronic genes. In this study, we investigate the kinetic mechanism underlying the programmed and spontaneous ribosome sliding. We show that the translation rate of gene 60 mRNA decreases as the ribosome approaches the take-off site, especially when the KKYK regulatory sequence in the nascent peptide reaches the constriction site in the ribosome exit tunnel. However, efficiency of bypassing increases when the ribosome traverses the gap quickly. With the non-coding gap exceeding the natural 50 nt, the processivity of sliding remains high up to 56 nt, but drops sharply beyond that due to the loss of mRNA elements support. Sliding efficiency is temperature-dependent; while temperature regulates the number of ribosomes initiating programmed bypassing, traversing the long gaps becomes increasingly unfavorable at lower temperatures. This data offers novel insights into the kinetic determinants of programmed and spontaneous ribosome sliding along the mRNA.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Ribossomos/metabolismo , Ribossomos/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/química , Cinética , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Regiões 3' não Traduzidas
4.
Viruses ; 16(4)2024 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-38675925

RESUMO

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.


Assuntos
Proteínas de Fusão gag-pol , Infecções por HIV , HIV-1 , Proteínas de Ligação a RNA , Humanos , Mudança da Fase de Leitura do Gene Ribossômico , Proteínas de Fusão gag-pol/genética , Proteínas de Fusão gag-pol/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Infecções por HIV/virologia , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , HIV-1/fisiologia , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Replicação Viral , Proteínas de Ligação a RNA/metabolismo
5.
PLoS Comput Biol ; 20(3): e1011918, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38442108

RESUMO

Processive enzymes like polymerases or ribosomes are often studied in bulk experiments by monitoring time-dependent signals, such as fluorescence time traces. However, due to biomolecular process stochasticity, ensemble signals may lack the distinct features of single-molecule signals. Here, we demonstrate that, under certain conditions, bulk signals from processive reactions can be decomposed to unveil hidden information about individual reaction steps. Using mRNA translation as a case study, we show that decomposing a noisy ensemble signal generated by the translation of mRNAs with more than a few codons is an ill-posed problem, addressable through Tikhonov regularization. We apply our method to the fluorescence signatures of in-vitro translated LepB mRNA and determine codon-position dependent translation rates and corresponding state-specific fluorescence intensities. We find a significant change in fluorescence intensity after the fourth and the fifth peptide bond formation, and show that both codon position and encoded amino acid have an effect on the elongation rate. This demonstrates that our approach enhances the information content extracted from bulk experiments, thereby expanding the range of these time- and cost-efficient methods.


Assuntos
Biossíntese de Proteínas , Ribossomos , Códon/genética , Códon/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/metabolismo , Fluorescência
6.
Mol Cell ; 84(4): 614-615, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38364778

RESUMO

Svetlov et al. identify the enzyme peptidyl-tRNA hydrolase as a ribosome-associated quality-control factor that promotes hydrolysis of the dislodged peptidyl-tRNA, which helps to recycle ribosomal subunits blocked by truncated nascent chains in bacteria.


Assuntos
Hidrolases de Éster Carboxílico , Ribossomos , Ribossomos/genética , Hidrolases de Éster Carboxílico/genética , Subunidades Ribossômicas , Bactérias
7.
Curr Opin Struct Biol ; 84: 102740, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38071940

RESUMO

During protein synthesis, the growing nascent peptide chain moves inside the polypeptide exit tunnel of the ribosome from the peptidyl transferase center towards the exit port where it emerges into the cytoplasm. The ribosome defines the unique energy landscape of the pioneering round of protein folding. The spatial confinement and the interactions of the nascent peptide with the tunnel walls facilitate formation of secondary structures, such as α-helices. The vectorial nature of protein folding inside the tunnel favors local intra- and inter-molecular interactions, thereby inducing cotranslational folding intermediates that do not form upon protein refolding in solution. Tertiary structures start to fold in the lower part of the tunnel, where interactions with the ribosome destabilize native protein folds. The present review summarizes the recent progress in understanding the driving forces of nascent protein folding inside the tunnel and at the surface of the ribosome.


Assuntos
Dobramento de Proteína , Ribossomos , Ribossomos/metabolismo , Biossíntese de Proteínas , Proteínas/metabolismo , Peptídeos/metabolismo
8.
J Mol Biol ; : 168384, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38065274

RESUMO

The mRNA coding sequence defines not only the amino acid sequence of the protein, but also the speed at which the ribosomes move along the mRNA while making the protein. The non-uniform local kinetics - denoted as translational rhythm - is similar among mRNAs coding for related protein folds. Deviations from this conserved rhythm can result in protein misfolding. In this review we summarize the experimental evidence demonstrating how local translation rates affect cotranslational protein folding, with the focus on the synonymous codons and patches of charged residues in the nascent peptide as best-studied examples. Alterations in nascent protein conformations due to disturbed translational rhythm can persist off the ribosome, as demonstrated by the effects of synonymous codon variants of several disease-related proteins. Charged amino acid patches in nascent chains also modulate translation and cotranslational protein folding, and can abrogate translation when placed at the N-terminus of the nascent peptide. During cotranslational folding, incomplete nascent chains navigate through a unique conformational landscape in which earlier intermediate states become inaccessible as the nascent peptide grows. Precisely tuned local translation rates, as well as interactions with the ribosome, guide the folding pathway towards the native structure, whereas deviations from the natural translation rhythm may favor pathways leading to trapped misfolded states. Deciphering the 'folding code' of the mRNA will contribute to understanding the diseases caused by protein misfolding and to rational protein design.

9.
ACS Chem Biol ; 18(12): 2441-2449, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37962075

RESUMO

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.


Assuntos
Ácidos Nucleicos , RNA , Epigênese Genética , Biologia
10.
Nat Commun ; 14(1): 4784, 2023 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-37553384

RESUMO

N6-methyladenosine (m6A) is an abundant, dynamic mRNA modification that regulates key steps of cellular mRNA metabolism. m6A in the mRNA coding regions inhibits translation elongation. Here, we show how m6A modulates decoding in the bacterial translation system using a combination of rapid kinetics, smFRET and single-particle cryo-EM. We show that, while the modification does not impair the initial binding of aminoacyl-tRNA to the ribosome, in the presence of m6A fewer ribosomes complete the decoding process due to the lower stability of the complexes and enhanced tRNA drop-off. The mRNA codon adopts a π-stacked codon conformation that is remodeled upon aminoacyl-tRNA binding. m6A does not exclude canonical codon-anticodon geometry, but favors alternative more dynamic conformations that are rejected by the ribosome. These results highlight how modifications outside the Watson-Crick edge can still interfere with codon-anticodon base pairing and complex recognition by the ribosome, thereby modulating the translational efficiency of modified mRNAs.


Assuntos
Anticódon , Biossíntese de Proteínas , Códon/genética , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
11.
Methods Enzymol ; 684: 39-70, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37230593

RESUMO

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.


Assuntos
Peptídeos , Ribossomos , Humanos , Ribossomos/metabolismo , Peptídeos/química , Escherichia coli/metabolismo , N-Formilmetionina/metabolismo , Bactérias/metabolismo , Amidoidrolases/química
12.
Annu Rev Biophys ; 52: 161-182, 2023 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-37159300

RESUMO

Faithful translation of messenger RNA (mRNA) into protein is essential to maintain protein homeostasis in the cell. Spontaneous translation errors are very rare due to stringent selection of cognate aminoacyl transfer RNAs (tRNAs) and the tight control of the mRNA reading frame by the ribosome. Recoding events, such as stop codon readthrough, frameshifting, and translational bypassing, reprogram the ribosome to make intentional mistakes and produce alternative proteins from the same mRNA. The hallmark of recoding is the change of ribosome dynamics. The signals for recoding are built into the mRNA, but their reading depends on the genetic makeup of the cell, resulting in cell-specific changes in expression programs. In this review, I discuss the mechanisms of canonical decoding and tRNA-mRNA translocation; describe alternative pathways leading to recoding; and identify the links among mRNA signals, ribosome dynamics, and recoding.


Assuntos
Proteostase , Ribossomos , RNA Mensageiro , Ribossomos/genética
13.
BBA Adv ; 3: 100074, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37082265

RESUMO

Elongation factor P (EF-P) and its eukaryotic homolog eIF5A are auxiliary translation factors that facilitate peptide bond formation when several sequential proline (Pro) residues are incorporated into the nascent chain. EF-P and eIF5A bind to the exit (E) site of the ribosome and contribute to favorable entropy of the reaction by stabilizing tRNA binding in the peptidyl transferase center of the ribosome. In most organisms, EF-P and eIF5A carry a posttranslational modification that is crucial for catalysis. The chemical nature of the modification varies between different groups of bacteria and between pro- and eukaryotes, making the EF-P-modification enzymes promising targets for antibiotic development. In this review, we summarize our knowledge of the structure and function of EF-P and eIF5A, describe their modification enzymes, and present an approach for potential drug screening aimed at EarP, an enzyme that is essential for EF-P modification in several pathogenic bacteria.

14.
Biol Chem ; 404(8-9): 755-767, 2023 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-37077160

RESUMO

In each round of translation elongation, the ribosome translocates along the mRNA by precisely one codon. Translocation is promoted by elongation factor G (EF-G) in bacteria (eEF2 in eukaryotes) and entails a number of precisely-timed large-scale structural rearrangements. As a rule, the movements of the ribosome, tRNAs, mRNA and EF-G are orchestrated to maintain the exact codon-wise step size. However, signals in the mRNA, as well as environmental cues, can change the timing and dynamics of the key rearrangements leading to recoding of the mRNA into production of trans-frame peptides from the same mRNA. In this review, we discuss recent advances on the mechanics of translocation and reading frame maintenance. Furthermore, we describe the mechanisms and biological relevance of non-canonical translocation pathways, such as hungry and programmed frameshifting and translational bypassing, and their link to disease and infection.


Assuntos
Fator G para Elongação de Peptídeos , Ribossomos , RNA Mensageiro/metabolismo , Fator G para Elongação de Peptídeos/genética , Ribossomos/genética , Ribossomos/metabolismo , Biossíntese de Proteínas/genética , Códon/análise , Códon/metabolismo , Fases de Leitura , RNA de Transferência/genética
15.
EMBO J ; 42(2): e112372, 2023 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-36472247

RESUMO

Protein synthesis is crucial for cell growth and survival yet one of the most energy-consuming cellular processes. How, then, do cells sustain protein synthesis under starvation conditions when energy is limited? To accelerate the translocation of mRNA-tRNAs through the ribosome, bacterial elongation factor G (EF-G) hydrolyzes energy-rich guanosine triphosphate (GTP) for every amino acid incorporated into a protein. Here, we identify an EF-G paralog-EF-G2-that supports translocation without hydrolyzing GTP in the gut commensal bacterium Bacteroides thetaiotaomicron. EF-G2's singular ability to sustain protein synthesis, albeit at slow rates, is crucial for bacterial gut colonization. EF-G2 is ~10-fold more abundant than canonical EF-G1 in bacteria harvested from murine ceca and, unlike EF-G1, specifically accumulates during carbon starvation. Moreover, we uncover a 26-residue region unique to EF-G2 that is essential for protein synthesis, EF-G2 dissociation from the ribosome, and responsible for the absence of GTPase activity. Our findings reveal how cells curb energy consumption while maintaining protein synthesis to advance fitness in nutrient-fluctuating environments.


Assuntos
Bacteroides , Fator G para Elongação de Peptídeos , Animais , Camundongos , Bacteroides/genética , Bacteroides/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólise , Fator G para Elongação de Peptídeos/genética , Fator G para Elongação de Peptídeos/química , Ribossomos/metabolismo , RNA de Transferência/metabolismo
16.
Sci Rep ; 12(1): 12848, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35896582

RESUMO

Fluorescence reporter groups are important tools to study the structure and dynamics of proteins. Genetic code reprogramming allows for cotranslational incorporation of non-canonical amino acids at any desired position. However, cotranslational incorporation of bulky fluorescence reporter groups is technically challenging and usually inefficient. Here we analyze the bottlenecks for the cotranslational incorporation of NBD-, BodipyFL- and Atto520-labeled Cys-tRNACys into a model protein using a reconstituted in-vitro translation system. We show that the modified Cys-tRNACys can be rejected during decoding due to the reduced ribosome selectivity for the modified aa-tRNA and the competition with native near-cognate aminoacyl-tRNAs. Accommodation of the modified Cys-tRNACys in the A site of the ribosome is also impaired, but can be rescued by one or several Gly residues at the positions -1 to -4 upstream of the incorporation site. The incorporation yield depends on the steric properties of the downstream residue and decreases with the distance from the protein N-terminus to the incorporation site. In addition to the full-length translation product, we find protein fragments corresponding to the truncated N-terminal peptide and the C-terminal fragment starting with a fluorescence-labeled Cys arising from a StopGo-like event due to a defect in peptide bond formation. The results are important for understanding the reasons for inefficient cotranslational protein labeling with bulky reporter groups and for designing new approaches to improve the yield of fluorescence-labeled protein.


Assuntos
Aminoácidos , RNA de Transferência de Cisteína , Aminoácidos/metabolismo , Escherichia coli/genética , Biossíntese de Proteínas , Proteínas/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Cisteína/genética , RNA de Transferência de Cisteína/metabolismo , Ribossomos/metabolismo
17.
Nat Commun ; 13(1): 4231, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35869111

RESUMO

When reading consecutive mRNA codons, ribosomes move by exactly one triplet at a time to synthesize a correct protein. Some mRNA tracks, called slippery sequences, are prone to ribosomal frameshifting, because the same tRNA can read both 0- and -1-frame codon. Using smFRET we show that during EF-G-catalyzed translocation on slippery sequences a fraction of ribosomes spontaneously switches from rapid, accurate translation to a slow, frameshifting-prone translocation mode where the movements of peptidyl- and deacylated tRNA become uncoupled. While deacylated tRNA translocates rapidly, pept-tRNA continues to fluctuate between chimeric and posttranslocation states, which slows down the re-locking of the small ribosomal subunit head domain. After rapid release of deacylated tRNA, pept-tRNA gains unconstrained access to the -1-frame triplet, resulting in slippage followed by recruitment of the -1-frame aa-tRNA into the A site. Our data show how altered choreography of tRNA and ribosome movements reduces the translation fidelity of ribosomes translocating in a slow mode.


Assuntos
Escherichia coli , Mudança da Fase de Leitura do Gene Ribossômico , Códon/genética , Códon/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mudança da Fase de Leitura do Gene Ribossômico/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genética , Ribossomos/metabolismo
18.
Viruses ; 14(7)2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35891432

RESUMO

The interferon-induced host cell protein shiftless (SFL) was reported to inhibit human immunodeficiency virus (HIV) infection by blocking the -1 programmed ribosomal frameshifting (-1PRF) required for expression of the Gag-Pol polyprotein. However, it is not clear how SFL inhibits -1PRF. To address this question, we focused on a 36 amino acids comprising region (termed required for antiviral activity (RAA)) that is essential for suppression of -1PRF and HIV infection and is missing from SFL short (SFLS), a splice variant of SFL with unknown function. Here, we confirm that SFL, but not SFLS, inhibits HIV -1PRF and show that inhibition is cell-type-independent. Mutagenic and biochemical analyses demonstrated that the RAA region is required for SFL self-interactions and confirmed that it is necessary for ribosome association and binding to the HIV RNA. Analysis of SFL mutants with six consecutive amino-acids-comprising deletions in the RAA region suggests effects on binding to the HIV RNA, complete inhibition of -1PRF, inhibition of Gag-Pol expression, and antiviral activity. In contrast, these amino acids did not affect SFL expression and were partially dispensable for SFL self-interactions and binding to the ribosome. Collectively, our results support the notion that SFL binds to the ribosome and the HIV RNA in order to block -1PRF and HIV infection, and suggest that the multimerization of SFL may be functionally important.


Assuntos
Infecções por HIV , Aminoácidos , Antivirais , Humanos , Mutagênicos , RNA
19.
Front Mol Biosci ; 9: 871121, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35573737

RESUMO

Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and - for polytopic membrane proteins - the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding.

20.
Nucleic Acids Res ; 50(9): 5282-5298, 2022 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-35489072

RESUMO

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Códon de Iniciação/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Humanos , Mamíferos/genética , Iniciação Traducional da Cadeia Peptídica , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA