Asunto(s)
Adenosina/metabolismo , Biosíntesis de Péptidos , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Adenosina/química , Catálisis , Concentración de Iones de Hidrógeno , Modelos Químicos , Protones , ARN Ribosómico/química , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/químicaRESUMEN
Approaches have been developed for the kinetic dissection of eukaryotic translation initiation in vitro using rabbit reticulocyte ribosomes and a crude preparation of initiation factors. These new approaches have allowed the kinetics of formation of the 43S and 80S ribosomal complexes to be followed and have substantially improved the ability to follow formation of the first peptide bond. The results suggest the existence of a new step on the initiation pathway that appears to require at least one additional factor and the hydrolysis of GTP and may prepare the 80S complex for the formation of the first peptide bond. The initial kinetic framework and methods developed herein will allow the properties of individual species along the initiation pathway to be probed further and will facilitate dissection of the mechanistic roles of individual translation factors and their interplay with RNA structural elements.
Asunto(s)
Biosíntesis de Proteínas/fisiología , Animales , Escherichia coli/metabolismo , Guanosina Trifosfato/metabolismo , Guanilil Imidodifosfato/metabolismo , Hidrólisis , Cinética , Modelos Biológicos , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/metabolismo , ARN de Transferencia de Metionina/metabolismo , Conejos , Reticulocitos/fisiología , Factores de TiempoRESUMEN
eIF4A is the archetypal member of the DEAD box family of proteins and has been proposed to use the energy from ATP hydrolysis to unwind structures in the 5'-untranslated regions of eukaryotic mRNAs during translation initiation. As a step toward understanding the mechanism of action of this class of enzymes, a minimal kinetic and thermodynamic framework for the RNA-activated ATPase function has been established for eIF4A. The enzyme's affinity for ssRNA is modulated by the binding of ATP.Mg2+ and ADP.Mg2+: the affinity of the E.ATP complex for ssRNA is approximately 40-fold higher than that of the E.ADP complex. The enzyme binds its substrates in a random manner; contrary to previous suggestions, neither ATP binding nor hydrolysis is required for binding of single-stranded RNA. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch that modulates the enzyme's structure and ssRNA affinity. The data presented in this and the following paper in this issue suggest that ATP binding and hydrolysis produce a cycle of conformational and RNA affinity changes in eIF4A. Such cycles may be used by DEAD box proteins to transduce the energy from ATP hydrolysis into physical work, thereby allowing each member of this family to rearrange its RNA or RNA.protein target.
Asunto(s)
Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , ARN/química , ARN/metabolismo , Nucleótidos de Adenina/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Reactivos de Enlaces Cruzados , Factor 4A Eucariótico de Iniciación , Humanos , Hidrólisis , Técnicas In Vitro , Cinética , Ratones , Datos de Secuencia Molecular , Conformación Proteica/efectos de la radiación , ARN/genética , ARN Nucleotidiltransferasas/química , ARN Nucleotidiltransferasas/metabolismo , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Termodinámica , Rayos UltravioletaRESUMEN
Limited proteolysis experiments have been carried out with the DEAD box protein eIF4A. The results suggest that there is a substantial conformational change in eIF4A upon binding single-stranded RNA. Binding of ADP induces conformational changes in the free enzyme and the enzyme.RNA complex, and binding of the ATP analogue AMP-PNP induces a conformational change in the enzyme.RNA complex. The presence or absence of the gamma-phosphate on the bound nucleotide acts as a switch, presumably via the Walker motifs, that mediates changes in protein conformation and, as described in the preceding paper in this issue, also mediates changes in RNA affinity. Thus, these results suggest that there is a series of changes in conformation and substrate affinity throughout the ATP hydrolysis reaction cycle. A model is proposed in which eIF4A and the eIF4A-like domains of the DEAD box proteins act as ATP-driven conformational switches or motors that produce movements or structural rearrangements of attached protein domains or associated proteins. These movements could then be used to rearrange RNA structures or RNA.protein complexes.
Asunto(s)
Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Nucleótidos de Adenina/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Tampones (Química) , Factor 4A Eucariótico de Iniciación , Hidrólisis , Técnicas In Vitro , Cinética , Ligandos , Ratones , Modelos Químicos , Datos de Secuencia Molecular , Factores de Iniciación de Péptidos/genética , Conformación Proteica , ARN/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , TripsinaRESUMEN
In Tom Stoppard's famous play [Rosencrantz and Guildenstern are Dead], the ill-fated heroes toss a coin 101 times. The first 100 times they do so the coin lands heads up. The chance of this happening is approximately 1 in 10(30), a sequence of events so rare that one might argue that it could only happen in such a delightful fiction. Similarly rare events, however, may underlie the origins of biological catalysis. What is the probability that an RNA, DNA, or protein molecule of a given random sequence will display a particular catalytic activity? The answer to this question determines whether a collection of such sequences, such as might result from prebiotic chemistry on the early earth, is extremely likely or unlikely to contain catalytically active molecules, and hence whether the origin of life itself is a virtually inevitable consequence of chemical laws or merely a bizarre fluke. The fact that a priori estimates of this probability, given by otherwise informed chemists and biologists, ranged from 10(-5) to 10(-50), inspired us to begin to address the question experimentally. As it turns out, the chance that a given random sequence RNA molecule will be able to catalyze an RNA polymerase-like phosphoryl transfer reaction is close to 1 in 10(13), rare enough, to be sure, but nevertheless in a range that is comfortably accessible by experiment. It is the purpose of this Account to describe the recent advances in combinatorial biochemistry that have made it possible for us to explore the abundance and diversity of catalysts existing in nucleic acid sequence space.
Asunto(s)
ADN , Evolución Molecular , Origen de la Vida , ARN Catalítico/química , Secuencia de Bases , Sitios de Unión , CatálisisRESUMEN
We have previously isolated a series of ribozymes with polynucleotide kinase activity [Lorsch, J.R., & Szostak, J.W. (1994) Nature 371, 31-36]. In order to learn how such newly evolved RNAs effect catalysis, we have determined a number of the kinetic and thermodynamic parameters for the reaction catalyzed by one of these ribozymes. This ribozyme, a class I polynucleotide kinase, catalyzes the transfer of the gamma-(thio)phosphate from ATP(-gamma S) to the 5'-hydroxyl of a 7-mer oligoribonucleotide. The kcat for the reaction with ATP-gamma S is 0.17 min-1 with a Km of approximately 3 mM. The Km for the oligoribonucleotide substrate 5'-HO-GGAACCU-3' is 2 microM, the same as the Kd for this substrate in the presence or absence of ATP-gamma S. Neither the binding of substrates nor the release of products is the rate-limiting step of the reaction. The binding of substrates and release of products appear to occur in a random fashion, with no synergy of binding between the ATP(-gamma S) and oligoribonucleotide substrates. The ribozyme binds the oligoribonucleotide substrate no more strongly than would be expected for the formation of a simple RNA-RNA duplex, suggesting that there are no tertiary contacts between the ribozyme and the RNA substrate. The oligoribonucleotide substrate binding site has been located, and the sequence specificity of the ribozyme could be altered by mutating this binding site. The ribozyme is specific for adenosine triphosphate substrates; GTP-gamma S reacts approximately 650-fold slower than ATP-gamma S. With ATP as the substrate, the Kms remain unchanged, but kcat decreases by a factor of 50, consistent with a rate-limiting chemical step occurring through a dissociative transition state. The pH independence (from pH 5.5 to 8.5) of kcat/Km and of the rate constant for the conversion of the ternary substrate complex into the ternary products complex is also consistent with a dissociative phosphoryl transfer mechanism. These results suggest that this newly evolved catalyst operates in a relatively simple manner, with independent substrate binding sites and without changing the mechanism of the underlying chemical reaction.
Asunto(s)
Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , ARN Catalítico/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sitios de Unión , Catálisis , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligorribonucleótidos/metabolismo , Polinucleótido 5'-Hidroxil-Quinasa/química , Polinucleótido 5'-Hidroxil-Quinasa/genética , ARN Catalítico/química , ARN Catalítico/genética , Especificidad por Sustrato , TermodinámicaRESUMEN
Avian myeloblastosis virus and Maloney murine leukemia virus RNase H-reverse transcriptases pause when they encounter a 2'-5' linkage or a 2'-thiophosphate in their template RNAs, but eventually read through these backbone modifications. Both reverse transcriptases pause after the 2'-5' linkage but before the 2'-thiophosphate. These results suggest that in the absence of precise information concerning the behavior of a given reverse transcriptase with respect to a particular lesion or modification, caution should be exercised in the interpretation of primer extension data that is being used to determine the existence of, or map the position of, a crosslink, site of chemical modification or non-standard linkage in an RNA template.
Asunto(s)
Fosfatos/química , ADN Polimerasa Dirigida por ARN/metabolismo , ARN/metabolismo , Virus de la Mieloblastosis Aviar/enzimología , Secuencia de Bases , ADN/biosíntesis , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/enzimología , Polinucleótido 5'-Hidroxil-Quinasa , ARN/síntesis química , ARN/química , Moldes GenéticosRESUMEN
We have isolated a large number of polynucleotide kinase ribozymes from a pool of RNA molecules consisting of an ATP-binding domain flanked by regions of random sequence. Different classes of kinases catalyse the transfer of the gamma-thiophosphate of ATP-gamma S to the 5'-hydroxyl or to internal 2'-hydroxyls. An engineered version of one class is able to catalyse the transfer of thiophosphate from ATP-gamma S to the 5'-hydroxyl of an exogenous oligoribonucleotide substrate with multiple turnover, thus acting as a true enzyme.
Asunto(s)
Evolución Biológica , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , ARN Catalítico/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Catálisis , Clonación Molecular , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Polinucleótido 5'-Hidroxil-Quinasa/clasificación , ARN/metabolismo , Selección Genética , Endonucleasas Específicas del ADN y ARN con un Solo Filamento , Tionucleótidos/metabolismoRESUMEN
RNA receptors (aptamers) capable of specifically binding cyanocobalamin (vitamin B12) have been isolated by in vitro selection from a pool of 5 x 10(14) RNAs of random sequence. After eight rounds of selection by affinity chromatography and enzymatic amplification, the pool was dominated by two sequences. The major sequence, comprising 60% of the pool, was studied further. It was found to bind vitamin B12 in solution with a dissociation constant (Kd) of approximately 320 +/- 90 nM and to bind cobinamide dicyanide with a Kd of 8.8 +/- 0.5 microM. The aptamer does not detectably bind adenosylcobalamin (coenzyme B12). The selection was conducted in 1 M LiCl, and binding is dependent on the presence of high concentrations of Li+ but independent of Mg2+. To define the binding site for cyanocobalamin, a second cyanocobalamin-binding selection was carried out using a pool of sequences derived from the major aptamer sequence randomized at a level of 30%. The sequence data from this selection revealed a 31-base highly conserved region, on the basis of which was synthesized a smaller aptamer of 35 nucleotides. This small aptamer binds cyanocobalamin in solution with a Kd of 88 +/- 19 nM and cobinamide dicyanide with a Kd of 20 +/- 9 microM. This aptamer has the highest affinity yet reported for a small molecule ligand. A number of covarying positions were found in the conserved region of the sequences from this second, mutagenized pool selection. On the basis of these data, an unusual pseudoknot secondary structure is proposed for the aptamer. Chemical modification protection experiments are consistent with this structure and have demonstrated that the RNA undergoes a conformational change upon binding its ligand. Possible contacts with the cyanocobalamin have also been mapped. A third selection was carried out in which the salt specificity of the aptamer was changed from LiCl to NaCl plus MgCl2. Sequence analysis of the final round pool of RNAs from this selection revealed several conserved changes from the original vitamin B12 aptamer sequence.
Asunto(s)
ARN/metabolismo , Vitamina B 12/metabolismo , Secuencia de Bases , Sitios de Unión , Cloruro de Litio , Cloruro de Magnesio , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , ARN/química , Cloruro de SodioRESUMEN
The July-August 1991 HBR presented "A New Compact for Owners and Directors," a set of principles for reconciling differences between owners and managers. In "Advice and Dissent: Rating the Corporate Governance Compact," a panel of three experts evaluates the Compact--and takes issue with its fundamental recommendation. Clifton R. Wharton, Jr., chairman and CEO of TIAA-CREF, describes how his organization brings delinquent managers and directors to task. Harvard Business School professor Jay W. Lorsch explains why strengthening the role of outside directors will develop more effective corporate control. And Lord Hanson, chairman of Hanson PLC, reaffirms the importance of maintaining a unitary board of directors and maximizing shareholder value.
Asunto(s)
Personal Administrativo/psicología , Comercio/organización & administración , Toma de Decisiones en la Organización , Consejo Directivo , Hospitales con Fines de Lucro/organización & administración , Humanos , Relaciones Interprofesionales , Propiedad , Estados UnidosRESUMEN
Universal theories do not always fit the situation in which they are used, and management practice is no exception. In fact, the difficulty in applying such behavioral science theories has been the interpretation that they are applicable to all situations. This author asks managers and academics alike to recognize that the easy way does not always work, that more theories should be developed to fit different situations, and that staffs should be educated in the theories and techniques that are available.