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1.
Mol Cell ; 75(5): 1007-1019.e5, 2019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31471187

RESUMO

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.


Assuntos
Escherichia coli/química , Conformação de Ácido Nucleico , Pinças Ópticas , RNA Bacteriano/química , RNA Mensageiro/química , Ribossomos/química , Escherichia coli/metabolismo , Fluorescência , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
2.
Proc Natl Acad Sci U S A ; 115(6): 1286-1291, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29351994

RESUMO

In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.


Assuntos
Algoritmos , RNA Polimerases Dirigidas por DNA/química , Pinças Ópticas , Pareamento de Bases , RNA Polimerases Dirigidas por DNA/genética , Difosfatos/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Cadeias de Markov
3.
Nucleic Acids Res ; 45(5): 2865-2874, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-27799473

RESUMO

Programmed -1 ribosomal frameshifting (-1PRF) is tightly regulated by messenger RNA (mRNA) sequences and structures in expressing two or more proteins with precise ratios from a single mRNA. Using single-molecule fluorescence resonance energy transfer (smFRET) between (Cy5)EF-G and (Cy3)tRNALys, we studied the translational elongation dynamics of -1PRF in the Escherichia coli dnaX gene, which contains three frameshifting signals: a slippery sequence (A AAA AAG), a Shine-Dalgarno (SD) sequence and a downstream hairpin. The frameshift promoting signals mostly impair the EF-G-catalyzed translocation step of the two tRNALys and the slippery codons from the A- and P- sites. The hairpin acts as a road block slowing the translocation rate. The upstream SD sequence together with the hairpin promotes dissociation of futile EF-G and thus causes multiple EF-G driven translocation attempts. A slippery sequence also helps dissociation of the EF-G by providing alternative base-pairing options. These results indicate that frameshifting takes place during the repetitive ribosomal conformational changes associated with EF-G dissociation upon unsuccessful translocation attempts of the second slippage codon from the A- to the P- sites.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Fator G para Elongação de Peptídeos/metabolismo , Proteínas de Bactérias/genética , Biocatálise , Códon , DNA Polimerase III/genética , Transferência Ressonante de Energia de Fluorescência , Mutação , Elongação Traducional da Cadeia Peptídica , RNA Mensageiro/química , RNA de Transferência de Lisina/metabolismo
4.
Science ; 348(6233): 457-60, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25908824

RESUMO

Protein synthesis rates can affect gene expression and the folding and activity of the translation product. Interactions between the nascent polypeptide and the ribosome exit tunnel represent one mode of regulating synthesis rates. The SecM protein arrests its own translation, and release of arrest at the translocon has been proposed to occur by mechanical force. Using optical tweezers, we demonstrate that arrest of SecM-stalled ribosomes can indeed be rescued by force alone and that the force needed to release stalling can be generated in vivo by a nascent chain folding near the ribosome tunnel exit. We formulate a kinetic model describing how a protein can regulate its own synthesis by the force generated during folding, tuning ribosome activity to structure acquisition by a nascent polypeptide.


Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Elongação Traducional da Cadeia Peptídica , Dobramento de Proteína , Ribossomos/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Técnicas In Vitro , Cinética , Fenômenos Mecânicos , Pinças Ópticas , Ribossomos/química
5.
Cell ; 160(5): 870-881, 2015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25703095

RESUMO

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.


Assuntos
Mutação da Fase de Leitura , Biossíntese de Proteínas , RNA Mensageiro/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , DNA Polimerase III/genética , Escherichia coli/metabolismo , Técnicas In Vitro , Espectrometria de Massas , Dados de Sequência Molecular
6.
Biopolymers ; 103(8): 424-31, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25269998

RESUMO

My personal view of ethical behavior as a scientific researcher in an academic environment is presented. I discuss the behavior of a graduate student, a postdoctoral, and a professor. Ethical behavior in teaching, choosing a research project, publishing papers, and obtaining a job is discussed.


Assuntos
Pesquisadores/ética
7.
Elife ; 3: e03406, 2014 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-25114092

RESUMO

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano-chemical cycle.


Assuntos
Códon/química , Biossíntese de Proteínas , RNA Mensageiro/química , RNA de Transferência/química , Ribossomos/química , Fenômenos Biomecânicos , Códon/metabolismo , Escherichia coli , Cinética , Movimento (Física) , Pinças Ópticas , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Termodinâmica
8.
Proc Natl Acad Sci U S A ; 111(15): 5538-43, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24706807

RESUMO

Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed -1 ribosomal frameshifting (-1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of -1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine-Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA(Lys) in the ribosomal peptidyl-tRNA-binding (P) site. We observed significantly slower elongation factor G (EF-G)-catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as -1PRF.


Assuntos
Escherichia coli/fisiologia , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Modelos Genéticos , Modelos Moleculares , Conformação Molecular , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/genética , DNA Polimerase III/genética , Escherichia coli/genética , Transferência Ressonante de Energia de Fluorescência , Ribossomos/fisiologia
9.
Annu Rev Biophys ; 43: 1-17, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24702008

RESUMO

Life at Berkeley for the past 57 years involved research on the thermodynamics, kinetics, and spectroscopic properties of RNA to better understand its structures, interactions, and functions. We (myself and all the graduate students and postdocs who shared in the fun) began with dinucleoside phosphates and slowly worked our way up to megadalton-sized RNA molecular motors. We used UV absorption, circular dichroism, circular intensity differential scattering, fluorescence, NMR, and single-molecule methods. We learned a lot and had fun doing it.


Assuntos
RNA/química , Termodinâmica , California , Dicroísmo Circular , História do Século XX , Cinética , Espectroscopia de Ressonância Magnética
11.
Biopolymers ; 99(12): 1147-66, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23722586

RESUMO

Translation of messenger RNA by a ribosome occurs three nucleotides at a time from start signal to stop. However, a frameshift means that some nucleotides are read twice or some are skipped, and the following sequence of amino acids is completely different from the sequence in the original frame. In some messenger RNAs, including viral RNAs, frameshifting is programmed with RNA signals to produce specific ratios of proteins vital to the replication of the organism. The mechanisms that cause frameshifting have been studied for many years, but there are no definitive conclusions. We review ribosome structure and dynamics in relation to frameshifting dynamics provided by classical ensemble studies, and by new single-molecule methods using optical tweezers and FRET.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Ribossomos , Sequência de Bases , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , RNA Viral , Ribossomos/química
12.
Proc Natl Acad Sci U S A ; 109(36): 14458-63, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22908248

RESUMO

The sequence and secondary structure of the 5'-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5'-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure ("stand-by site") before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.


Assuntos
Modelos Biológicos , Conformação de Ácido Nucleico , Biossíntese de Proteínas/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Escherichia coli , Pinças Ópticas , Reação em Cadeia da Polimerase , Biossíntese de Proteínas/genética , RNA de Cadeia Dupla/química , RNA Mensageiro/química , Proteínas Ribossômicas/química
13.
Science ; 334(6063): 1723-7, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22194581

RESUMO

Proteins are synthesized by the ribosome and generally must fold to become functionally active. Although it is commonly assumed that the ribosome affects the folding process, this idea has been extremely difficult to demonstrate. We have developed an experimental system to investigate the folding of single ribosome-bound stalled nascent polypeptides with optical tweezers. In T4 lysozyme, synthesized in a reconstituted in vitro translation system, the ribosome slows the formation of stable tertiary interactions and the attainment of the native state relative to the free protein. Incomplete T4 lysozyme polypeptides misfold and aggregate when free in solution, but they remain folding-competent near the ribosomal surface. Altogether, our results suggest that the ribosome not only decodes the genetic information and synthesizes polypeptides, but also promotes efficient de novo attainment of the native state.


Assuntos
Muramidase/química , Dobramento de Proteína , Ribossomos/metabolismo , Proteínas Virais/química , Bacteriófago T4 , Teorema de Bayes , Cadeias de Markov , Muramidase/biossíntese , Muramidase/metabolismo , Pinças Ópticas , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Termodinâmica , Proteínas Virais/biossíntese , Proteínas Virais/metabolismo
14.
Science ; 333(6050): 1746-9, 2011 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-21940894

RESUMO

Nonhexameric helicases use adenosine triphosphate (ATP) to unzip base pairs in double-stranded nucleic acids (dsNAs). Studies have suggested that these helicases unzip dsNAs in single-base pair increments, consuming one ATP molecule per base pair, but direct evidence for this mechanism is lacking. We used optical tweezers to follow the unwinding of double-stranded RNA by the hepatitis C virus NS3 helicase. Single-base pair steps by NS3 were observed, along with nascent nucleotide release that was asynchronous with base pair opening. Asynchronous release of nascent nucleotides rationalizes various observations of its dsNA unwinding and may be used to coordinate the translocation speed of NS3 along the RNA during viral replication.


Assuntos
Hepacivirus/enzimologia , RNA Helicases/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Pareamento de Bases , Cinética , Modelos Biológicos , Conformação de Ácido Nucleico , Pinças Ópticas , RNA de Cadeia Dupla/química , RNA Viral/química
15.
Nature ; 475(7354): 118-21, 2011 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-21734708

RESUMO

The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.


Assuntos
Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Mensageiro/química , RNA Mensageiro/genética , Ribossomos/metabolismo , Pareamento de Bases , Sequência de Bases , Códon/genética , Sequência Rica em GC/genética , Transcriptase Reversa do HIV/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Pinças Ópticas , Elongação Traducional da Cadeia Peptídica , RNA Helicases/química , RNA Helicases/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/enzimologia , Termodinâmica
16.
Genes Dev ; 25(12): 1205-31, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21685361

RESUMO

The last 15 years have witnessed the development of tools that allow the observation and manipulation of single molecules. The rapidly expanding application of these technologies for investigating biological systems of ever-increasing complexity is revolutionizing our ability to probe the mechanisms of biological reactions. Here, we compare the mechanistic information available from single-molecule experiments with the information typically obtained from ensemble studies and show how these two experimental approaches interface with each other. We next present a basic overview of the toolkit for observing and manipulating biology one molecule at a time. We close by presenting a case study demonstrating the impact that single-molecule approaches have had on our understanding of one of life's most fundamental biochemical reactions: the translation of a messenger RNA into its encoded protein by the ribosome.


Assuntos
Biologia Molecular , Animais , Humanos , Biologia Molecular/instrumentação , Biologia Molecular/métodos , Biologia Molecular/tendências , Biossíntese de Proteínas , Ribossomos/metabolismo , Espectrometria de Fluorescência , Análise Espectral , Fatores de Tempo
17.
Cold Spring Harb Perspect Biol ; 2(11): a003624, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20739416

RESUMO

Much of the dynamics information is lost in bulk measurements because of the population averaging. Single-molecule methods measure one molecule at a time; they provide knowledge not obtainable by other means. In this article, we review the application of the two most widely used single-molecule methods--fluorescence resonance energy transfer (FRET) and force versus extension measurements--to several RNA reactions. First, we discuss folding/unfolding studies on a hairpin ribozyme that revealed multiple conformations of the RNA with distinct kinetics, and on a series of RNA pseudoknots, whose mechanical stabilities were found to show a strong correlation with their frameshifting efficiency during translation. We also discuss several RNA-related molecular motors. Single-molecule experiments revealed detailed mechanisms for the interaction of HIV reverse transcriptase and nucleic acid helicases (NS3 and RIG-1) with their substrates. Optical tweezers studies showed that translation of a single messenger RNA by a ribosome occurs by successive translocation-and-pause cycles. Single-molecule FRET experiments yielded important information on ribosome conformational changes and tRNA dynamics during translation. Overall, single-molecule experiments have been very valuable for understanding RNA reactions.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Pinças Ópticas , RNA/química , Ribossomos/química , Cinética
18.
Proc Natl Acad Sci U S A ; 106(31): 12706-11, 2009 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-19628688

RESUMO

Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/química
19.
Phys Biol ; 6(2): 025006, 2009 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-19571367

RESUMO

In the cell, proteins are synthesized by ribosomes in a multi-step process called translation. The ribosome translocates along the messenger RNA to read the codons that encode the amino acid sequence of a protein. Elongation factors, including EF-G and EF-Tu, are used to catalyze the process. Recently, we have shown that translation can be followed at the single-molecule level using optical tweezers; this technique allows us to study the kinetics of translation by measuring the lifetime the ribosome spends at each codon. Here, we analyze the data from single-molecule experiments and fit the data with simple kinetic models. We also simulate the translation kinetics based on a multi-step mechanism from ensemble kinetic measurements. The mean lifetimes from the simulation were consistent with our experimental single-molecule measurements. We found that the calculated lifetime distributions were fit in general by equations with up to five rate-determining steps. Two rate-determining steps were only obtained at low concentrations of elongation factors. These analyses can be used to design new single-molecule experiments to better understand the kinetics and mechanism of translation.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Códon , Simulação por Computador , Cinética , Modelos Biológicos , Modelos Moleculares , Pinças Ópticas , Fator G para Elongação de Peptídeos/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , RNA Mensageiro/genética , Ribossomos/genética , Thermus thermophilus/metabolismo
20.
J Mol Biol ; 386(5): 1343-56, 2009 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-19452632

RESUMO

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg 2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 +/- 4 to 46 +/- 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of deltaX(++), which is the distance from the folded structure to the unfolding transition state. The deltaX(++) of Mal is approximately 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg 2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg 2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.


Assuntos
HIV-1/metabolismo , Magnésio/química , Potássio/química , RNA Viral/química , Cátions Bivalentes , Cátions Monovalentes , Dimerização , Cloreto de Magnésio/química , Conformação de Ácido Nucleico , Pinças Ópticas , Cloreto de Potássio/química , Dobramento de Proteína
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