RESUMO
A set of nuclear mutants of C. reinhardtii were identified that specifically lack translation of the chloroplast-encoded psbA mRNA, which encodes the photosystem II reaction center polypeptide D1. Two of these mutants are deficient in the 47-kD member (RB47) of the psbA RNA-binding complex, which has previously been identified both genetically and biochemically as a putative translational activator of the chloroplast psbA mRNA. RB47 is a member of the poly(A)-binding protein family, and binds with high affinity and specificity to the 5' untranslated region of the psbA mRNA. The results presented here confirm RB47's role as a message-specific translational activator in the chloroplast, and bring together genetic and biochemical data to form a cohesive model for light- activated translational regulation in the chloroplast.
Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Expressão Gênica , Luz , Modelos Biológicos , Mutagênese Insercional , Mutação , Complexo de Proteína do Fotossistema II , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismoRESUMO
High-affinity binding of a set of proteins with specificity for the 5' untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message. We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family. Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation. In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein. RB47 expressed and purified from Escherichia coli binds to the psbA 5' UTR with similar specificity and affinity as RB47 isolated from C. reinhardtii chloroplasts. The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA. These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Proteínas de Ligação a Poli(A) , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.
Assuntos
Núcleo Celular/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Biossíntese de Proteínas/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Acetatos/metabolismo , Animais , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , Polirribossomos/metabolismoRESUMO
Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine-Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message-specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.
Assuntos
Chlamydomonas reinhardtii/genética , Conformação de Ácido Nucleico , Complexo de Proteínas do Centro de Reação Fotossintética/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/genética , Animais , Sequência de Bases , Chlamydomonas reinhardtii/metabolismo , Chlamydomonas reinhardtii/efeitos da radiação , Análise Mutacional de DNA , Escuridão , Regulação da Expressão Gênica/efeitos da radiação , Luz , Dados de Sequência Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema II , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Muscle contraction consists of a cyclical interaction between myosin and actin driven by the concomitant hydrolysis of adenosine triphosphate (ATP). A model for the rigor complex of F actin and the myosin head was obtained by combining the molecular structures of the individual proteins with the low-resolution electron density maps of the complex derived by cryo-electron microscopy and image analysis. The spatial relation between the ATP binding pocket on myosin and the major contact area on actin suggests a working hypothesis for the crossbridge cycle that is consistent with previous independent structural and biochemical studies.