RESUMEN

The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.

Asunto(s)

Antimetabolitos/farmacología , Tiofenos/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Antimetabolitos/síntesis química , Antimetabolitos/química , Sitios de Unión , Humanos , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/química , Activador de Plasminógeno de Tipo Uroquinasa/fisiología

RESUMEN

The abundance of structural data available for retroviral proteases affords a unique opportunity to investigate structure activity relationships. Our approach attempts to genetically engineer an HIV (human immunodeficiency virus)-1 protease that is functionally equivalent to the HIV-2 and the SIV (simian immunodeficiency virus) enzymes and conversely to engineer an HIV-2 protease that is functionally equivalent to the HIV-1 enzyme. For this purpose, the HIV-2 and SIV proteases were cloned and characterized in an Escherichia coli (E. coli) assay system along with 33 engineered HIV-1 and HIV-2 enzymes. The results of these experiments show that a relatively large S1 or S1' subsite volume, which is likely determined by the conformation of the 80's loop (residues 78 to 85), is necessary to fully accommodate the HIV-1 protease specificity site AETF*YCDG (the asterisk indicates the location scissile bond) during productive binding.

Asunto(s)

Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Lentivirus de los Primates/enzimología , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Escherichia coli/genética , Proteínas de Fusión gag-pol/metabolismo , Productos del Gen gag/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , Especificidad por Sustrato

RESUMEN

A heterologous substrate assay for the human immunodeficiency virus type 1 (HIV-1) protease has been engineered in Escherichia coli. This assay detects the activity of the HIV-1 protease within intact bacterial cells and does not require biochemical purification of either the enzyme or the substrate. For this assay, nine HIV-1 protease specificity sites were genetically engineered into a heterologous protein (galactokinase) and the relative processing of these substrates by the wild-type and a substituted HIV-1 protease was determined. The results from these experiments revealed that the activity of the HIV-1 protease in the engineered heterologous substrate assay is consistent with previously reported in vitro assays and in vivo observations as well as a proposed catalytic specificity model.

Asunto(s)

Escherichia coli/enzimología , Proteasa del VIH/química , Escherichia coli/genética , Galactoquinasa/genética , Galactoquinasa/metabolismo , Vectores Genéticos , Proteasa del VIH/genética , Proteasa del VIH/metabolismo , VIH-1/enzimología , Plásmidos , Especificidad por Sustrato/genética

RESUMEN

The herpes simplex virus type 1 protease is expressed as an 80,000-dalton polypeptide, encoded within the 635-amino acid open reading frame of the UL26 gene. The two known protein substrates for this enzyme are the protease itself and the capsid assembly protein ICP35 (Liu, F., and Roizman, B. (1991) J. Virol. 65, 5149-5156). In this report we describe the use of a rapid and quantitative assay for characterizing the protease. The assay uses a glutathione S-transferase fusion protein containing the COOH-terminal cleavage site of ICP35 as the substrate (GST-56). The protease consists of N0, the NH2-terminal 247 amino acid catalytic domain of the UL26 gene product, also expressed as a GST fusion protein. Upon cleavage with N0, a single 25-mer peptide is released from GST-56, which is soluble in trichloroacetic acid. Using this assay, the protease displayed a pH optimum between 7 and 9 but most importantly had an absolute requirement for high concentrations of an antichaeotrophic agent. Strong salting out salts such as Na2SO4 and KPO4 (> or = 1 M) stimulated activity, whereas NaCl and KCl had no effect. The degree of stimulation by 1.25 M Na2SO4 and KPO4 were 100-150- and 200-300-fold, respectively. Using the fluorescent probe 1-anilino-8-naphthalene sulfonate, the protease was shown to bind the dye in the presence of 1.25 M Na2SO4 or KPO4, but not at low ionic strength or in the presence of 1.25 or 2.2 M NaCl. This binding was most likely at the protease active site because a high affinity cleavage site peptide, but not a control peptide, could displace the dye. In addition to cleaving GST-56, the herpes simplex virus type I protease also cleaved the purified 56-mer peptide. Circular dichroism and NMR spectroscopy showed the peptide to be primarily random coil under physiological conditions, suggesting that antichaeotrophic agents affect the conformation of the substrate as well as the protease.

Asunto(s)

Sales (Química)/farmacología , Serina Endopeptidasas/metabolismo , Proteínas Virales , Acetatos/farmacología , Secuencia de Aminoácidos , Cloruros/farmacología , Activación Enzimática , Genes Virales , Glutatión Transferasa/biosíntesis , Glutatión Transferasa/metabolismo , Herpesvirus Humano 1/enzimología , Herpesvirus Humano 1/genética , Cinética , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Fosfatos/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/aislamiento & purificación , Espectrometría de Fluorescencia , Especificidad por Sustrato , Sulfatos/farmacología

RESUMEN

The herpes simplex virus type 1 protease and related proteins are involved in the assembly of viral capsids. The protease encoded by the UL26 gene can process itself and its substrate ICP35, encoded by the UL26.5 gene. To better understand the functions of the protease in infected cells, we have isolated a complementing cell line (BMS-MG22) and constructed and characterized a null UL26 mutant virus, m100. The mutant virus failed to grow on Vero cells and required a complementing cell line for its propagation, confirming that the UL26 gene product is essential for viral growth. Phenotypic analysis of m100 shows that (i) normal amounts of the c and d forms of ICP35 were produced, but they failed to be processed to the cleaved forms, e and f; (ii) viral DNA replication of the mutant proceeded at near wild-type levels, but DNA was not processed to unit length or encapsidated; (iii) capsid structures were observed in thin sections of m100-infected Vero cells by electron microscopy, but assembly of VP5 into hexons of the capsid structure was conformationally altered; and (iv) nuclear localizations of the protease and ICP35 are independent of each other, and the function(s) of Na, at least in part, is to direct the catalytic domain N(o) to the nucleus.

Asunto(s)

Cápside/biosíntesis , Endopeptidasas/fisiología , Herpesvirus Humano 1/crecimiento & desarrollo , Proteínas Virales , Animales , Secuencia de Bases , Cápside/genética , Cápside/metabolismo , Proteínas de la Cápside , Línea Celular , Células Clonales , Cricetinae , Cartilla de ADN/genética , ADN Viral/genética , Endopeptidasas/genética , Genes Virales , Prueba de Complementación Genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis , Fenotipo , Procesamiento Proteico-Postraduccional , Temperatura , Células Vero

RESUMEN

The herpes simplex virus type 1 (HSV-1) protease is cleaved at two autoprocessing sites during viral maturation, one of which shares amino acid identity with its substrate, ICP35. Similar autoprocessing sites have been observed within other members of the Herpesviridae. Introduction of point mutations within the autoprocessing sites of the HSV-1 protease indicated that specificity resides within the P4-P1' region of the cleavage sites.

Asunto(s)

Endopeptidasas/metabolismo , Herpesvirus Humano 1/enzimología , Secuencia de Aminoácidos , Análisis Mutacional de ADN , Endopeptidasas/genética , Datos de Secuencia Molecular , Mutación Puntual , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Proteínas Virales/metabolismo

RESUMEN

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.

Asunto(s)

Cápside/metabolismo , Endopeptidasas/metabolismo , Simplexvirus/enzimología , Proteínas Virales , Alanina , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , Clonación Molecular , Endopeptidasas/biosíntesis , Endopeptidasas/aislamiento & purificación , Escherichia coli/genética , Genes Virales , Mutagénesis Sitio-Dirigida , Plásmidos , Mutación Puntual , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Serina , Simplexvirus/genética , Trombina/metabolismo

RESUMEN

HIV-1 protease contains two identical, conformationally mobile loops, known as flaps, which form in part the binding pockets for substrates and inhibitors. We have constructed a site-specific mutant of the protease in which residues Phe-53 and Phe-153 at the end of the flaps have been mutated to Trp residues, in order to incorporate a specific fluorescent probe to monitor conformational changes upon the binding of an inhibitor. The Phe53Trp (F53W) mutant of HIV-1 protease was expressed in Escherichia coli and purified from bacterial lysates. Analysis of the purified mutant protease demonstrated that its kinetic properties were highly similar to those of the wild-type protease. While binding of a potent peptide-analogue inhibitor (Ki = 9 nM) to the wild-type enzyme led to no change in protein fluorescence, a 5-8% increase in fluorescence was observed with the F53W mutant, indicating an enhancement of the Trp fluorescence due to flap movement upon inhibitor binding. Investigation of the kinetics of the F53W protease-inhibitor binding by stopped-flow spectrofluorometry revealed a rapid increase in protein fluorescence upon formation of the enzyme-inhibitor complex. These data were consistent with a one-step mechanistic model of inhibitor binding in which flap movement was concomitant with inhibitor binding, from which respective rate constants of association and dissociation of 2.5 x 10(6) M-1 s-1 and 0.023 s-1 were obtained.

Asunto(s)

Inhibidores de la Proteasa del VIH/metabolismo , Proteasa del VIH/química , Proteasa del VIH/metabolismo , VIH-1/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Proteasa del VIH/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Péptidos/metabolismo , Fenilalanina/genética , Conformación Proteica , Espectrometría de Fluorescencia , Triptófano/genética , Triptófano/metabolismo , Difracción de Rayos X

RESUMEN

Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35). The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease. The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus. The fusion protease underwent autoproteolytic cleavage at two distinct sites. The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data. The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis. Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease. The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.

Asunto(s)

Endopeptidasas/química , Fragmentos de Péptidos/química , Simplexvirus/enzimología , Proteínas Virales , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Sitios de Unión , Clonación Molecular , Secuencia Conservada , ADN Viral/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Expresión Génica , Immunoblotting , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia , Homología de Secuencia de Aminoácido , Proteína Estafilocócica A/genética

RESUMEN

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.

Asunto(s)

Endopeptidasas/metabolismo , Escherichia coli/genética , Simplexvirus/enzimología , Proteínas Virales/metabolismo , Western Blotting , Clonación Molecular , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Genes Virales , Cinética , Plásmidos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Simplexvirus/genética , Especificidad por Sustrato , Factores de Tiempo

RESUMEN

The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0

Asunto(s)

Ácido Aspártico Endopeptidasas/química , Proteasa del VIH/química , VIH-1/enzimología , Virus de la Inmunodeficiencia de los Simios/enzimología , Ácido Aspártico Endopeptidasas/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Proteasa del VIH/efectos de los fármacos , Calor , Modelos Químicos , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Termodinámica , Ultracentrifugación , Urea/farmacología

RESUMEN

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.

Asunto(s)

Endopeptidasas/aislamiento & purificación , Proteasa del VIH/aislamiento & purificación , VIH-1/enzimología , Proteínas de los Retroviridae/aislamiento & purificación , Virus de la Inmunodeficiencia de los Simios/enzimología , Secuencia de Aminoácidos , Animales , Endopeptidasas/química , Endopeptidasas/clasificación , Proteasa del VIH/clasificación , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH , VIH-1/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/farmacología , Conformación Proteica , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/clasificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas de los Retroviridae/antagonistas & inhibidores , Proteínas de los Retroviridae/clasificación , Proteínas de los Retroviridae/genética , Virus de la Inmunodeficiencia de los Simios/fisiología , Especificidad por Sustrato

RESUMEN

RNase mapping experiments show that the bovine growth hormone (bGH) poly(A) region forms an extensive hairpin loop. Mutants were prepared to change poly(A) region pre-mRNA structure and cleavage site efficiency without altering necessary sequences. An inverted repeat which includes the poly(A) cleavage site was created by insertion of a linker upstream of the poly(A) region to compete with any wild-type secondary structure. RNA mapping analyses show alterations in the nuclease accessibility of this mutant at the natural site of cleavage. This mutant shows a 75% drop in relative reporter gene expression at the steady-state protein and RNA levels. When the linker is inserted as a direct repeat, expression is equivalent to wildtype levels. To show that transcription was not terminated by the inverted repeat, the SV40 late poly(A) region was inserted downstream. These mutants show restored expression and processing at the downstream site. Our experiments reveal that the conformation of the poly(A) site pre-mRNA is important in mediating efficient cleavage-polyadenylation.

Asunto(s)

Hormona del Crecimiento/genética , Poli A , Precursores del ARN/ultraestructura , Procesamiento Postranscripcional del ARN , ARN Mensajero , Animales , Secuencia de Bases , Bovinos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poli A/biosíntesis , Poli A/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/biosíntesis

Asunto(s)

Escherichia coli/metabolismo , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Centrifugación por Gradiente de Densidad/métodos , Indicadores y Reactivos , Cinética , ARN Mensajero/aislamiento & purificación , ARN Ribosómico/aislamiento & purificación , Técnica de Dilución de Radioisótopos , Proteínas Ribosómicas/aislamiento & purificación , Radioisótopos de Azufre , Termodinámica , Transcripción Genética

RESUMEN

Expression of the four ribosomal proteins from the Escherichia coli alpha operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the alpha mRNA. Using a filter binding assay and alpha mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the approximately 100-base leader sequence. We have extended this work to include fragments of the alpha leader with six different 5' end points and four different 3' end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of approximately 2 microM-1. Regions of weak interactions are located in the 22 nucleotides 5' and the 70 nucleotides 3' to this core; they increase the S4 affinity to approximately 13 microM-1. Studies of S4-alpha mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with delta log+ K/delta log [K+] approximately 4 in most potassium salts. With KCl and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 mM enhances binding by a factor of 4, with no further effects up to 20 mM. About five Mg2+ are taken up by the complex with an average binding constant of approximately 600 M-1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with alpha mRNA extending beyond the initiation codon; S4 binding to the alpha leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.

Asunto(s)

Escherichia coli/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Ribosómicas/metabolismo , Termodinámica , Secuencia de Bases , Cinética , Magnesio/metabolismo , Operón , ARN Bacteriano/metabolismo

RESUMEN

The secondary structure of the Escherichia coli alpha mRNA leader sequence has been determined using nucleases specific for single- or double-stranded RNA. Three different length alpha RNA fragments were studied at 0 degrees C and 37 degrees C. A very stable eight base-pair helix forms upstream from the ribosome initiation site, defining a 29 base loop. There is evidence for base-pairing between nucleotides within this loop and for a "pseudo-knot" interaction of some loop bases with nucleotides just 3' to the initiation codon, forming a region of complex structure. A weak helix also pairs sequences near the 5' terminus of the alpha mRNA with bases near the Shine-Dalgarno sequence. Affinity constants for the translational repressor S4 binding different length alpha mRNA fragments indicate that most of the S4 recognition features must be contained within the main helix and hairpin regions. Binding of S4 to the alpha mRNA alters the structure of the 29 base hairpin region, and probably melts the weak pairing between the 5' and 3' termini of the leader. The pseudo-knot structure and the conformational changes associated with it provide a link between the structures of the S4 binding site and the ribosome binding site. The alpha mRNA may therefore play an active role in mediating translational repression.

Asunto(s)

Escherichia coli/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Ribosómicas/metabolismo , Autorradiografía , Secuencia de Bases , Sitios de Unión , Modelos Moleculares , Conformación de Ácido Nucleico , Operón , ARN Bacteriano/metabolismo

RESUMEN

The Escherichia coli ribosomal protein S4 is known to repress translation of its own gene and several other ribosomal protein (r-protein) genes in the alpha operon as part of a general mechanism coordinating the levels of rRNA and r-protein synthesis. Using a filter binding assay and RNA transcripts prepared in vitro, we have detected and quantitated specific interactions between S4 and alpha mRNA fragments. The main results are the following: Only the alpha mRNA leader is required for specific recognition, with a small fraction of the binding free energy derived from sequences at the ribosome initiation site. 16S rRNA and alpha mRNA compete for binding to S4 with about the same affinity (approximately equal to 2 X 10(7) M-1), suggesting that S4 utilizes the same recognition features in each RNA. Nonspecific binding of S4 to tRNA or other mRNA sequences is strongly salt dependent, while the specific S4-alpha mRNA affinity is nearly independent of salt. At physiological salt concentrations the nonspecific S4-RNA affinity (10(5)-10(6) M-1) is large enough to strongly buffer the free S4 concentration in vivo.

Asunto(s)

Operón , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Escherichia coli/metabolismo , Cinética , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Proteínas Ribosómicas/aislamiento & purificación , Ribosomas/metabolismo , Transcripción Genética