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1.
Nat Commun ; 12(1): 4644, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330903

RESUMO

Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-G•GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.


Assuntos
Mudança da Fase de Leitura do Gene Ribossômico/genética , Elongação Traducional da Cadeia Peptídica/genética , Fator G para Elongação de Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Ribossomos/genética , Biocatálise , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Fator G para Elongação de Peptídeos/química , Fator G para Elongação de Peptídeos/metabolismo , Conformação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Ribossomos/metabolismo , Ribossomos/ultraestrutura , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
2.
Nucleic Acids Res ; 45(19): 11043-11055, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28977553

RESUMO

In prokaryotes, RNA polymerase and ribosomes can bind concurrently to the same RNA transcript, leading to the functional coupling of transcription and translation. The interactions between RNA polymerase and ribosomes are crucial for the coordination of transcription with translation. Here, we report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits. RNA polymerase and ribosomes form a one-to-one complex with a micromolar dissociation constant. The formation of the complex is modulated by the conformational and functional states of RNA polymerase and the ribosome. The binding interface on the large ribosomal subunit is buried by the small subunit during protein synthesis, whereas that on the small subunit remains solvent-accessible. The RNA polymerase binding site on the ribosome includes that of the isolated small ribosomal subunit. This direct interaction between RNA polymerase and ribosomes may contribute to the coupling of transcription to translation.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas/metabolismo , Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Cinética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Subunidades Ribossômicas/química , Subunidades Ribossômicas/genética
3.
RNA Biol ; 13(5): 477-85, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26950678

RESUMO

We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Deficiências do Desenvolvimento/genética , Epilepsia Resistente a Medicamentos/genética , Mutação , Ribonuclease P/metabolismo , Análise de Sequência de DNA/métodos , Criança , Exoma , Genes Ligados ao Cromossomo X , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo
4.
Int J Mol Sci ; 16(7): 14866-83, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26140378

RESUMO

Native tRNAs often contain post-transcriptional modifications to the wobble position to expand the capacity of reading the genetic code. Some of these modifications, due to the ability to confer imperfect codon-anticodon pairing at the wobble position, can induce a high propensity for tRNA to shift into alternative reading frames. An example is the native UGG isoacceptor of E. coli tRNAPro whose wobble nucleotide U34 is post-transcriptionally modified to cmo5U34 to read all four proline codons (5'-CCA, 5'-CCC, 5'-CCG, and 5'-CCU). Because the pairing of the modified anticodon to CCC codon is particularly weak relative to CCA and CCG codons, this tRNA can readily shift into both the +1 and +2-frame on the slippery mRNA sequence CCC-CG. We show that the shift to the +2-frame is more dominant, driven by the higher stability of the codon-anticodon pairing at the wobble position. Kinetic analysis suggests that both types of shifts can occur during stalling of the tRNA in a post-translocation complex or during translocation from the A to the P-site. Importantly, while the +1-frame post complex is active for peptidyl transfer, the +2-frame complex is a poor peptidyl donor. Together with our recent work, we draw a mechanistic distinction between +1 and +2-frameshifts, showing that while the +1-shifts are suppressed by the additional post-transcriptionally modified m1G37 nucleotide in the anticodon loop, the +2-shifts are suppressed by the ribosome, supporting a role of the ribosome in the overall quality control of reading-frame maintenance.


Assuntos
Mutação da Fase de Leitura , RNA de Transferência de Prolina/genética , Pareamento de Bases , Códon/genética , Escherichia coli/genética , RNA Mensageiro/genética
5.
Nat Commun ; 6: 7226, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-26009254

RESUMO

Maintaining the translational reading frame poses difficulty for the ribosome. Slippery mRNA sequences such as CC[C/U]-[C/U], read by isoacceptors of tRNA(Pro), are highly prone to +1 frameshift (+1FS) errors. Here we show that +1FS errors occur by two mechanisms, a slow mechanism when tRNA(Pro) is stalled in the P-site next to an empty A-site and a fast mechanism during translocation of tRNA(Pro) into the P-site. Suppression of +1FS errors requires the m(1)G37 methylation of tRNA(Pro) on the 3' side of the anticodon and the translation factor EF-P. Importantly, both m(1)G37 and EF-P show the strongest suppression effect when CC[C/U]-[C/U] are placed at the second codon of a reading frame. This work demonstrates that maintaining the reading frame immediately after the initiation of translation by the ribosome is an essential aspect of protein synthesis.


Assuntos
Mutação da Fase de Leitura , Biossíntese de Proteínas , Fases de Leitura , Ribossomos/metabolismo , Escherichia coli , Fatores de Alongamento de Peptídeos/metabolismo
6.
RNA ; 15(10): 1827-36, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19696158

RESUMO

The universally conserved CCA sequence is present at the 3' terminal 74-76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic parameters. Specifically, the kinetics of C74 and C75 addition is controlled by the chemistry of nucleotidyl transfer, whereas the kinetics of A76 addition is controlled by a prechemistry conformational transition of the active site. In multiple turnover condition, all three steps are controlled by slow product release, indicating enzyme processivity from one addition to the next. However, the processivity decreases as the enzyme progresses to complete the CCA synthesis. Together, these results suggest the existence of a network of diverse kinetic parameters that determines the overall rate of CCA addition for tRNA maturation.


Assuntos
RNA de Transferência/química , Sequência de Bases , Catálise , Domínio Catalítico , Cinética , Conformação de Ácido Nucleico
7.
Biochemistry ; 45(22): 6978-86, 2006 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-16734433

RESUMO

The existence of secondary structure in long single-stranded DNA and RNA is a serious obstacle to the practical use of short oligonucleotide probes (<20-mers). Here, we show that replication of a highly structured DNA in the presence of a unique set of dNTP analogues leads to synthesis of daughter DNA with a significantly reduced level of secondary structure. This replicated DNA, composed of 2-aminoadenine, 2-thiothymine, 7-deazaguanine, and cytosine bases, was readily accessible to tiled 8-mer LNA and 15-mer DNA probes, whereas an unmodified version of the same DNA was inaccessible. Importantly, while the base analogues enhanced probe-target stability, they did not significantly reduce the specificity of base pairing. The availability of structure-free DNA targets should facilitate the use of short oligonucleotide probes and promote development of generic oligonucleotide microarrays.


Assuntos
Replicação do DNA , DNA/química , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Sondas de DNA/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química
8.
RNA ; 11(9): 1441-7, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16120834

RESUMO

The propensity of RNA to fold into higher-order structures poses a major barrier to the use of short probes (<15 nucleotides) by preventing their accessibility. Introduction of the pseudo-complementary bases 2-aminoadenine (nA) and 2-thiouracil (sU) and the destabilizing base 7-deazaguanine (cG) into RNA provides a partial solution to this problem. While complementary in hydrogen bonding groups, nA and sU cannot form a stable base pair due to steric hindrance, and are thus pseudo-complementary. Each, however, recognizes the regular T/U and A complements, allowing pairing with oligonucleotides. Short pseudo-complementary RNAs can be prepared by in vitro transcription. Relative to standard transcripts, the modified transcripts possess reduced secondary structure and increased accessibility to short (8-mer) probes in the locked nucleic acid (LNA) configuration. They also hybridize to complementary probes with increased specificity and thermostability. Practical application of this strategy to oligonucleotide-based hybridization assays will require engineering of RNA polymerase for more efficient utilization of pseudo-complementary nucleoside triphosphates.


Assuntos
Pareamento de Bases/fisiologia , Conformação de Ácido Nucleico , Oligonucleotídeos/metabolismo , Sondas RNA/metabolismo , RNA/metabolismo , 2-Aminopurina/análogos & derivados , 2-Aminopurina/metabolismo , Guanina/análogos & derivados , Guanina/metabolismo , Hipoxantina/metabolismo , Hibridização de Ácido Nucleico , Temperatura , Tiouracila/metabolismo
9.
Biochemistry ; 43(31): 10224-36, 2004 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-15287750

RESUMO

Secondary structure in RNA targets is a significant barrier to short DNA probes. However, when such targets are the end product of an in vitro amplification scheme, it is possible to carry out transcription in the presence of nucleoside triphosphate analogues that reduce secondary structure of the RNA without impairing subsequent hybridization. Here we show that nucleoside triphosphates of 2-aminoadenine (nA) and 2-thiouracil (sU) are taken up by T7 RNA polymerase and that the resulting RNA possesses reduced secondary structure and improved accessibility to DNA probes. The hybridization properties of short RNA transcripts were studied using a new gel mobility shift assay from which melting temperatures were determined. RNA hairpins that contained nA and sU were able to hybridize to DNA probes under conditions where the unmodified hairpins did not. DNA-RNA hybrids that contained nA and sU in the RNA strand exhibited enhanced specificity, increased stability, and greater equality of base pairing strength than the same hybrids without modifications. Substitution of guanine (G) with inosine (I) further reduced secondary structure, but RNA with this base hybridized nonselectively. The high stability of nA-T and A-sU base pairs in DNA-RNA hybrids, combined with the destabilizing effect of the nA-sU couple in RNA targets, accounts for the improved hybridization properties. These results suggest that incorporation of nA and sU during in vitro transcription is a promising strategy for enhancing the performance of oligomeric DNA probes with an RNA target.


Assuntos
2-Aminopurina/análogos & derivados , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/metabolismo , 2-Aminopurina/metabolismo , Bacteriófago T7/enzimologia , Composição de Bases , Pareamento Incorreto de Bases , Pareamento de Bases , Sondas de DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Estabilidade Enzimática , Temperatura Alta , Inosina/metabolismo , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , Hibridização de Ácido Nucleico , Tiouracila/metabolismo , Proteínas Virais
10.
Biochemistry ; 42(9): 2643-55, 2003 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-12614159

RESUMO

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Oligonucleotídeos/química , Recombinases Rec A/química , Recombinação Genética , Trifosfato de Adenosina/química , DNA de Cadeia Simples/química , Eletroforese em Gel de Poliacrilamida , Globinas/química , Globinas/genética , Humanos , Canamicina Quinase/química , Canamicina Quinase/genética , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico , Sondas RNA/química , Homologia de Sequência do Ácido Nucleico
11.
Biomol Eng ; 20(1): 7-20, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12485680

RESUMO

Studies involving the alteration of DNA sequences by modified single-stranded oligonucleotides in vitro and in vivo have revealed potential applications for functional genomics. Repair of a replacement, deletion, or insertion mutation has already been achieved with molecules having lengths between 25 and 74 bases. But, other vector parameters still remain to be explored. Here, the position of the single base in the vector directing the alteration was examined and the optimal site was found to be at or near the center of the vector. If that position is staggered 3' or 5', the frequencies of gene repair in vitro decreases. The potential of a single vector to direct two nucleotide changes at a specific site in a target sequence was also examined. Both targeted bases are corrected together at the same frequency if the sites are separated by three bases, but conversion linkage decreases precipitously when the distance is expanded to 15 and 27 nucleotides, respectively. These results suggest that single oligonucleotides can be used to direct nucleotide exchange at two independent sites, a reaction characteristic that may be useful for many genomics applications.


Assuntos
Cinamatos , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Vetores Genéticos/genética , Higromicina B/análogos & derivados , Mutagênese Sítio-Dirigida/genética , Oligonucleotídeos/genética , Pareamento Incorreto de Bases/genética , Pareamento de Bases/genética , Sequência de Bases , Células Cultivadas , DNA Fúngico/genética , Marcação de Genes/métodos , Engenharia Genética/métodos , Higromicina B/metabolismo , Dados de Sequência Molecular , Controle de Qualidade , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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