RESUMEN
Genetic and genomic discovery is revolutionizing medicine at an extraordinary pace, leading to a better understanding of disease and improved treatments for patients. This advanced pace of discovery presents an urgency to expand medical school curricula to include genetic and genomic testing (including pharmacogenomics), and integration of genomic medicine into clinical practice. Consequently, organizations and healthcare authorities have charged medical schools with training future physicians to be competent in their knowledge of genomic implementation.
Asunto(s)
Curriculum/tendencias , Farmacogenética/educación , Facultades de Medicina/tendencias , Descubrimiento de Drogas , Genómica/educación , HumanosRESUMEN
Labeled peptides synthesized by core facilities are frequently used by researchers for following trafficking of a peptide, for binding studies, to determine substrate specificity, and for receptor cross-linking studies.The membership of the Association of Biomolecular Resource Facilities was asked to participate in a study focusing on synthesis of a biotin-labeled peptide, and it was suggested that a new strategy, using Rink amide 4-methylbenzhydrylamine resin coupled with Fmoc-Lys(Dde)-OH, be used.This strategy can be used for addition of a variety of labels other than biotin and should prove useful to core facilities. Comparison of the new strategy to other strategies was performed. Biotin labeling has long been assumed to be routine and specific. Despite the assumed routine nature of synthesizing biotinylated peptides, 9 of the 34 samples submitted did not contain any of the correct product. Although synthesis using Fmoc-Lys(Dde)-OH plus biotin generally gave the highest yields, other approaches also yielded a high percentage of the correct product.Therefore, the various strategies are generally comparable. The major advantage of this new approach is that other labels such as fluorescein, dansyl groups, methyl coumarin, and potentially fluorophores and quenchers used for fluorescence resonance energy transfer (FRET) can be directly incorporated into peptides.
RESUMEN
Nucleotide sequence of Xanthomonas oryzae pv. oryzae (Xoo) DNA from pSM-A1 was determined and sequence analysis revealed an ORF with high homology to RecA proteins. Expression analysis using an anti-RecA antibody demonstrates that MMS treatment induces recA in Xanthomonas strains but not in an Escherichia coli harbouring cloned Xoo recA. This indicates the existence of a recA regulatory mechanism in Xanthomonas that is not function in E. coli. In Xoo, recA was highly induced by treatments with chemical mutagens, UV and peroxides, while superoxides, a thiol agent, a heavy metal and heat shock were not inducers. The increased amount of RecA induced by H2O2 or MMS treatments were due to increased transcription of recA. recA showed no growth phase or starvation regulation. The pattern of recA regulation in Xoo could play important roles in stress survival in the environment and during plant-microbe interactions.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Rec A Recombinasas/genética , Xanthomonas/genética , Proteínas Bacterianas/genética , Northern Blotting , Daño del ADN , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Mutágenos/farmacología , Estrés Oxidativo/genética , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Rec A Recombinasas/análisis , Análisis de Secuencia de ADN , Rayos Ultravioleta , Xanthomonas/efectos de los fármacos , Xanthomonas/crecimiento & desarrolloRESUMEN
Cells of Bacillus subtilis can enter a natural physiological state, termed competence, that is permissive for uptake of DNA from the surrounding medium. In the B. subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, followed by intracellular processing that may ultimately lead to productive infection. Previous investigations have shown that transfecting DNA is usually far less infectious (on a molar basis) than is the DNA injected by phage particles; this result is apparently due to inactivating events suffered by transfecting DNA during its metabolism by competent cells. Earlier studies also demonstrated that, in some cases, the infectivity of transfecting DNA can be increased by ultraviolet (UV) irradiation of the competent cells prior to transfection, or by cotransfection of UV-irradiated heterologous DNAs; collectively, these phenomena have been termed transfection enhancement (TE). We propose here that some transfecting B. subtilis phage DNAs are attacked by a novel host DNA repair system, and that TE reflects inhibition of this by a competing substrate in UV-irradiated DNA. In support of this model, we show that UV-DNA cotransfection leads to a reduced rate of intracellular endonucleolytic breakdown of transfecting DNA. We also demonstrate that TE displays marked specificity of a kind frequently observed for repair enzymes. Thus, phages that contain hydroxymethyl uracil (HMU), but not thymine, in their genomes are susceptible to this process. In addition, we show that the photoproduct(s) in UV-irradiated DNA that produces TE by cotransfection is specific, and is not uracil, a pyrimidine dimer, thymine glycol, HMU, or a substrate for the E. coli thymine glycol DNA N-glycosylase. This photoproduct is derivable from thymine or HMU. The implications of these results are discussed.
Asunto(s)
Bacillus subtilis/genética , ADN Glicosilasas , Reparación del ADN/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/efectos de la radiación , Desoxirribonucleasa (Dímero de Pirimidina) , Proteínas de Escherichia coli , Transfección/métodos , Bacillus subtilis/efectos de la radiación , Bacteriófagos/genética , Bacteriófagos/efectos de la radiación , Composición de Base , Reparación del ADN/efectos de la radiación , ADN Bacteriano/genética , Endodesoxirribonucleasas/metabolismo , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , N-Glicosil Hidrolasas/efectos de la radiación , Pentoxil (Uracilo)/análogos & derivados , Pentoxil (Uracilo)/metabolismo , Pentoxil (Uracilo)/efectos de la radiación , Pirimidinas/metabolismo , Pirimidinas/efectos de la radiación , Especificidad por Sustrato , Timina/análogos & derivados , Timina/metabolismo , Timina/efectos de la radiación , Rayos Ultravioleta , Uracil-ADN GlicosidasaRESUMEN
In the Bacillus subtilis genetic system, transfection refers to uptake of isolated bacteriophage DNA by competent host cells, sometimes followed by productive cell infection. Previous studies have shown that ultraviolet (UV)-irradiation of the competent host cells, or cotransfection of UV-irradiated heterologous DNA, can increase the efficiency of transfection in some cases; these latter two phenomena have been called transfection enhancement (TE). In an accompanying paper, we show that TE is apparently confined to the B. subtilis phages that contain hydroxymethyluracil (HMU) in their DNA, and that the photoproduct in UV-irradiated DNA that mediates TE is specific, and different than the pyrimidine dimer, thymine glycol, uracil, or HMU. We also show that TE is due to reduced intracellular endonucleolytic attack of transfecting DNA. Based on this DNA base and nucleolytic specificity, we hypothesized that TE reflects the incidental action of a host DNA repair system on transfecting HMU phage DNA. In continuing these studies, we show here that duplex infecting HMU phage DNA is apparently inactivated by this same putative repair system when phage protein synthesis is blocked. We find, too, that this inactivation of infecting HMU phage DNA can be inhibited by UV-irradiated DNA, and that this process has a similar DNA base specificity as for TE. The survival of infecting HMU phage DNA is dependent on host DNA polymerase activity. We can detect specific DNA synthesis consistent with formation of repair patches when inactivation of infecting HMU phage DNA is ongoing, but not when it is inhibited by the presence of UV DNA or by allowing phage gene expression. Each of these results is consistent with the hypothesis that TE reflects the action of a novel DNA repair pathway. We show that a candidate TE-associated enzymatic activity can be detected in cell free extracts of uninfected, but not HMU phage-infected, B. subtilis cells. Correspondingly, the extracts of phage-infected cells appear to contain a diffusible factor that acts as an inhibitor of this host enzyme.
Asunto(s)
Bacillus subtilis/genética , Reparación del ADN/genética , ADN Bacteriano/metabolismo , ADN Bacteriano/efectos de la radiación , Bacillus subtilis/efectos de la radiación , Bacillus subtilis/virología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/efectos de la radiación , Bacteriófagos/genética , Bacteriófagos/efectos de la radiación , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , ADN Bacteriano/efectos de los fármacos , ADN Viral/biosíntesis , ADN Viral/efectos de la radiación , ADN Polimerasa Dirigida por ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/genética , Pentoxil (Uracilo)/análogos & derivados , Pentoxil (Uracilo)/metabolismo , Pentoxil (Uracilo)/efectos de la radiación , Polietilenglicoles/farmacología , Especificidad por Sustrato , Transfección/métodos , Rayos UltravioletaRESUMEN
Lens-specific expression of the mouse alpha A-crystallin gene is regulated at the level of transcription. Here, we have studied the role of the PE1 region, which contains the TATA box (-31/-26) and the immediately adjacent PE1B sequence (-25/-12), in transcriptional regulation. Deletions within either the TATA box or PE1B sequence eliminated promoter activity in transfected lens cells. Surprisingly, these deletions did not eliminate lens-specific promoter activity of the transgene of transgenic mice. Transcription of the transgene with a TATA-deleted promoter initiated at multiple sites in the lenses of the transgenic mice. Footprint analysis revealed that the entire PE1 region was protected by nuclear extracts prepared from lens cells which express the alpha A-crystallin gene and from fibroblasts which do not express the gene. The -37/+3 region formed three specific EMSA complexes using lens cell nuclear extracts, while a similar but much less intense pattern was observed when a fibroblast nuclear extract was used. Competition experiments indicated that these complexes were not due to the binding of TBP to the TATA box, but rather to the binding of other nuclear proteins to the PE1B -25/-19 region. A series of co-transfection competition studies in vivo also suggested the functional importance of proteins binding in the -25/-19 region. The PE1B protein-DNA interactions appear to be conserved in the chicken, rodent and human alpha A-crystallin gene as well as within the alpha A- and alpha B-crystallin genes in the mouse. Our findings indicate that the PE1B region is important for mouse alpha A-crystallin promoter activity; the proximity of this site to the TATA box raises the possibility for cooperativity or competition between TBP and PE1B-bound proteins.
Asunto(s)
Cristalinas/genética , Proteínas de Unión al ADN/metabolismo , Cristalino/metabolismo , Regiones Promotoras Genéticas/genética , Transcripción Genética/genética , Animales , Secuencia de Bases , Unión Competitiva , Extractos Celulares/química , Línea Celular , Núcleo Celular/química , Secuencia Conservada , ADN/metabolismo , Células Epiteliales , Cristalino/citología , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia/fisiología , TATA Box/genéticaRESUMEN
The dissociation constant (KD) for the complex of the intermediate dienol (2) and the D38N mutant of 3-oxo-delta 5-steroid isomerase (D38N.2) has been determined for the isomerization of 5-androstene-3,17-dione (1). KD for D38N.2 is pH-dependent, with values of 6 nM at pH 6.9, 51 nM at pH 5.8, and 59 nM at pH 5.2. These values of KD are used to estimate the pH-independent dissociation constant (0.7 +/- 0.3 microM) for the complex of dienol and wild-type (WT) enzyme. The internal equilibrium constant (Kint = 0.3 +/- 0.2) for the interconversion of bound substrate (WT.1) and bound intermediate (WT.2) was then calculated for WT using its KD, the values for the external equilibrium constant for 1<-->2, and the dissociation constant of the enzyme substrate complex (KS). The dissociation constant (KD) for the complex of equilenin (4) with WT, D38E, and D38N enzymes was also determined at pH values from 4 to 7. For the complex of 4 with D38N (D38N.4), KD is pH-dependent with an apparent pKa of about 4.5, whereas KD for both WT.4 and D38E.4 is pH-independent. These values are used to give two additional estimates of the internal equilibrium constant for WT (Kint = 0.5 and 0.01). Analysis of these results in terms of Marcus formalism leads to the conclusion that the primary function of the enzyme is to decrease the thermodynamic barrier to formation of the intermediate by lowering delta Gzero by about 10 kcal/mol. In contrast, the intrinsic free energy of activation (delta G++int) is only decreased by about 3 kcal/mol. These results are discussed in terms of competing theories of enzymatic enolization.
Asunto(s)
Mutagénesis Sitio-Dirigida , Esteroide Isomerasas/química , Catálisis , Concentración de Iones de Hidrógeno , Cinética , Pseudomonas/enzimología , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Esteroide Isomerasas/genética , Esteroide Isomerasas/metabolismoRESUMEN
The recA gene from the bacterium Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen, was cloned based on its ability to complement DNA repair defects of Escherichia coli recA- mutants. The Xoo recA was localized to a 1.3-kb Sau3AI-XhoI fragment and, when cloned into pBR322, specifies increased methylmethanesulfonate and mitomycin C resistance to E. coli recA mutants and allows lambda red- gam- to plaque on an E. coli recA- host. An E. coli recA- strain harboring a plasmid containing the Xoo recA-like gene was shown to produce a 40-kDa protein which cross-reacted with an anti-E. coli RecA antibody. A similar molecular mass protein to RecA has been detected in several Xanthomonas pathovars using an anti-E. coli RecA antibody. Furthermore, the cloned Xoo recA was shown to hybridize to genomic DNA from various Xanthomonas pathovars, but not to genomic DNA from other bacteria species under high-stringency hybridization conditions. These results indicate the isolation of the Xoo recA gene.
Asunto(s)
Rec A Recombinasas/genética , Xanthomonas/genética , Bacteriófago lambda/fisiología , Southern Blotting , Western Blotting , Clonación Molecular , Escherichia coli/efectos de los fármacos , Prueba de Complementación Genética , Metilmetanosulfonato/farmacología , Mitomicina/farmacología , Rec A Recombinasas/aislamiento & purificación , Rec A Recombinasas/metabolismo , Especificidad de la Especie , Ensayo de Placa ViralRESUMEN
Induction of cat-86 translation results from the stalling of a ribosome at a discrete location in the leader region of the transcript. Stalling destabilizes an adjacent region of secondary structure that sequesters the cat-86 ribosome binding site, thereby activating cat-86 translation. Two well characterized antibiotics, chloramphenicol and erythromycin, induce cat-86 by stalling a ribosome at the appropriate leader site. Here we demonstrate differences between the two antibiotics with respect to induction. First, induction by chloramphenicol is dependent on nucleotides in the leader sequence that are different from those necessary for erythromycin induction. Second, variants of Bacillus subtilis that are chloramphenicol resistant because of chromosome mutations permit cat-86 induction by chloramphenicol, whereas erythromycin-resistance host mutations block or greatly reduce cat-86 induction by erythromycin. Third, selected strains of B. subtilis bearing alterations in proteins of the 50S ribosomal subunit interfere with cat-86 induction by chloramphenicol, yet these strains are chloramphenicol sensitive. Lastly, induction by chloramphenicol is not reversed by removal of the antibiotic whereas erythromycin induction is reversible. The data indicate that chloramphenicol induction results from an effect of the drug that is not identical to its role as a general inhibitor of ribosome elongation. Induction by erythromycin, on the other hand, could not be distinguished from its antibiotic activity.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Bacillus subtilis/efectos de los fármacos , Proteínas Bacterianas/genética , Cloranfenicol O-Acetiltransferasa/genética , Resistencia al Cloranfenicol/genética , Inducción Enzimática/efectos de los fármacos , Eritromicina/farmacología , Conformación de Ácido Nucleico , Nucleósidos de Pirimidina/farmacología , ARN Bacteriano/genética , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas Ribosómicas/genética , Ribosomas/efectos de los fármacosRESUMEN
The induction of cat-86 by chloramphenicol has been proposed to follow the translational attenuation model. In the absence of inducer, the cat-86 gene is transcribed but remains phenotypically unexpressed because the transcripts sequester the ribosome binding site for the cat coding sequence in a stable stem-loop structure, preventing translation initiation. The translational attenuation model proposes that the natural inducer, chloramphenicol, stalls a ribosome in the leader region of cat transcripts, which causes localized melting of the downstream stem-loop structure, allowing initiation of translation of the cat-86 coding sequence. Although it is established that ribosome stalling in the cat-86 leader can induce translation of the coding sequence, several subsequent steps predicted by the model remain to be experimentally confirmed. As a consequence, the present evidence for cat-86 regulation can also be explained by two other potential control devices, ribosome hopping and translational frameshifting. Here we describe experiments designed to determine whether the alternatives to translational attenuation regulate cat-86. The results obtained are inconsistent with both competing models and are consistent with predictions made by the translational attenuation model.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Biosíntesis de Proteínas , Ribosomas/metabolismo , Bacillus subtilis/genética , Secuencia de Bases , Western Blotting , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/metabolismo , ARN Bacteriano/metabolismoRESUMEN
The translational attenuation regulatory model suggests a mechanism that can explain the induction of cat-86 by chloramphenicol (Cm). In this model, Cm serves to stall a ribosome at a specific site in a leader region of cat-86 transcripts. The stalled ribosome is thought to destabilize a downstream region of RNA secondary structure that normally sequesters the cat-86 ribosome-binding site (RBS-3). Three mutations in codon 4 of the cat-86 leader have been identified which result in constitutive cat expression. Each of the three mutations generates a likely -10 promoter sequence in the leader. Twenty nucleotides (nt) upstream is the wild-type sequence, 5'-TTGAAA, which differs from the consensus sigA -35 domain by only a single nt. The transcription start point from the resulting mutant promoter is within the DNA region that normally specifies the RNA secondary structure that sequesters cat-86 RBS-3. Thus, the basis for the constitutive phenotype is the absence of the RNA secondary structure in the transcripts driven by the promoter generated through mutagenesis of leader codon 4.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Codón/genética , Regulación Bacteriana de la Expresión Génica/genética , Plásmidos/genética , Transcripción Genética/genética , Secuencia de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Secuencia de Bases , Cloranfenicol/farmacología , Resistencia al Cloranfenicol , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismoRESUMEN
Replacement of cat-86 codon 7 or 144 with the UGA codon permitted the gene to confer chloramphenicol resistance in wild-type Bacillus subtilis. UAA replacements of the same codons resulted in a chloramphenicol-sensitive phenotype in wild-type B. subtilis and a chloramphenicol-resistant phenotype in suppressor-positive strains. N-terminal sequencing showed that UGA at codon 7 was decoded as tryptophan in wild-type cells, at an efficiency of about 6%.
Asunto(s)
Bacillus subtilis/genética , Codón/genética , Triptófano , Secuencia de Bases , Resistencia al Cloranfenicol/genética , Mapeo Cromosómico , Supresión Genética , Transformación BacterianaRESUMEN
Inducible cat and erm genes are regulated by translational attenuation. In this regulatory model, gene activation results from chloramphenicol- or erythromycin-dependent stalling of a ribosome at a precise site in the leader region of cat or erm transcripts. The stalled ribosome is believed to destabilize a downstream region of RNA secondary structure that sequesters the ribosome-binding site for the cat or erm coding sequence. Here we show that the ribosome stall sites in cat and erm leader mRNAs, designated crb and erb, respectively, are largely complementary to an internal sequence in 16S rRNA of Bacillus subtilis. A tetracycline resistance gene that is likely regulated by translational attenuation also contains a sequence in its leader mRNA, trb, which is complementary to a sequence in 16S rRNA that overlaps with the crb and erb complements. An in vivo assay is described which is designed to test whether 16S rRNA of a translating ribosome can interact with the crb sequence in mRNA in an inducer-dependent reaction. The assay compares the growth rate of cells expressing crb-86 with the growth rate of cells lacking crb-86 in the presence of subinhibitory levels of inducers of cat-86, chloramphenicol, fluorothiamphenicol, amicetin, or erythromycin. Under these conditions, crb-86 retarded growth. Deletion of the crb-86 sequence, insertion of ochre mutations into crb-86, or synonymous codon changes in crb-86 that decreased its complementarity with 16S rRNA all eliminated from detection inducer-dependent growth retardation. Lincomycin, a ribosomally targeted antibiotic that is not an inducer of cat-86, failed to selectively retard the growth of cells expressing crb-86. We suggest that cat-86 inducers enable the crb-86 sequence in mRNA to base pair with 16S rRNA of translating ribosome. When the base pairing is extensive, as with crb-86, ribosomes become transiently trapped on crb and are temporarily withdrawn from protein synthesis to the extent that growth rate declines. Site-specific positioning of an antibiotic-stalled ribosome is a hallmark of the translational attenuation model. The proposed rRNA-mRNA interaction may precisely position the ribosome on the stall site and perhaps contributes to stabilizing the ribosome leader mRNA complex.
Asunto(s)
Bacillus subtilis/genética , Señales de Clasificación de Proteína/genética , ARN Mensajero/genética , ARN Ribosómico 16S/genética , Ribosomas/metabolismo , Bacillus subtilis/crecimiento & desarrollo , Secuencia de Bases , Cloranfenicol/farmacología , Cloranfenicol O-Acetiltransferasa/genética , Deleción Cromosómica , Genes Bacterianos/efectos de los fármacos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Plásmidos , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
The cat-86 gene specifies chloramphenicol acetyltransferase (CAT). The cat-86 start codon is UUG, although related genes have AUG as the start codon. Changing the start codon to AUG increased expression of cat-86 by 36% in Bacillus subtilis. Changing the start codon to GUG and CUG decreased expression to 65% and 30%, respectively, of the level obtained when AUG was the start codon. CUG has not been previously shown to function as a start codon in B. subtilis. N-terminal sequencing of purified CAT protein specified by the CUG mutant, revealed that CUG was indeed the start codon and specified methionine. The gene xylE, which specifies catechol 2,3-dioxygenase, has AUG as its start codon. Changing the start codon for xylE to CUG decreased expression by 98%. However, when the ribosome-binding site sequence for xylE was optimized and the spacing between it and the start codon was increased to 8 nucleotides, xylE activity increased to 13% of the activity observed for AUG. CUG did not function efficiently as a start codon for cat-86 in Escherichia coli. These data suggest conditions under which CUG can function, with modest efficiency, as a start codon in B. subtilis.
Asunto(s)
Bacillus subtilis/genética , Cloranfenicol O-Acetiltransferasa/genética , Codón/genética , Genes Bacterianos , Mutagénesis Sitio-Dirigida , Bacillus subtilis/enzimología , Secuencia de Bases , Calorimetría , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Biosíntesis de ProteínasRESUMEN
Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol/farmacología , Codón , Señales de Clasificación de Proteína/genética , ARN Mensajero , Ribosomas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Ribosomas/efectos de los fármacos , Homología de Secuencia de Ácido NucleicoRESUMEN
The mutation sup-3 in Bacillus subtilis suppresses ochre (TAA) mutations at each of three codons in the 5' end of the cat-86 coding sequence. The suppressor is shown to insert lysine at ochre codons. The efficiency of suppression by sup-3 is about 15%, as determined by changing a cat-86 Lys codon (codon 12) to an ochre codon and measuring the level of CAT in the suppressor-containing strain. The results obtained are discussed in light of previous observations that ochre mutations at cat leader codons 2 and 3 can be phenotypically suppressed by sup-3, whereas ochre mutations at leader codons 4 and 5 cannot. Translation of the cat leader is essential to inducible expression of cat. Our data support the interpretation that the nature of amino acids 2 through 5 of the leader peptide contributes to determining whether chloramphenicol can stall a ribosome in the leader, which in turn leads to induction of cat expression.
Asunto(s)
Bacillus subtilis/genética , Cloranfenicol O-Acetiltransferasa/genética , Regulación Bacteriana de la Expresión Génica , Supresión Genética , Secuencia de Aminoácidos , Secuencia de Bases , Codón , Genes Bacterianos , Lisina , Datos de Secuencia Molecular , Biosíntesis de ProteínasRESUMEN
The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.
Asunto(s)
Aminoácidos/metabolismo , Bacillus subtilis/genética , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol/farmacología , Mutación , Secuencia de Aminoácidos , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/enzimología , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , Inducción Enzimática , Genes , Genes Bacterianos , Genes Reguladores , Cinética , Datos de Secuencia MolecularRESUMEN
Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.
Asunto(s)
Acetiltransferasas/genética , Bacillus subtilis/genética , Regulación de la Expresión Génica/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos , Antibacterianos/farmacología , Bacillus subtilis/enzimología , Secuencia de Bases , Cloranfenicol/farmacología , Cloranfenicol O-Acetiltransferasa , Codón/genética , Genes Bacterianos , Datos de Secuencia Molecular , Mutación , Plásmidos , Señales de Clasificación de Proteína/genética , Nucleósidos de Pirimidina/farmacología , RibosomasRESUMEN
The plasmid gene cat-86 specifies chloramphenicol-inducible chloramphenicol acetyltransferase in Bacillus subtilis. Induction by the antibiotic is primarily due to activation of the translation of cat-86-encoded mRNA. It has been suggested that the inducer stalls ribosomes at a discrete location in the leader region of cat-86 mRNA, which causes the destabilization of a downstream RNA secondary structure that normally sequesters the cat-86 ribosome binding site. It is the destabilization of this RNA secondary structure that permits translation of the cat-86 coding sequence. In the present report, we show that ribosomes that were stalled in the cat-86 leader by starvation of host cells for the amino acid specified by leader codon 6 induced gene expression to a level above that detected when cells were starved for the amino acids specified by leader codons 7 and 8. Starvation for amino acids specified by leader codons 3, 4, or 5 failed to activate cat-86 expression. These results indicate that the stalled ribosome that is most active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To determine if chloramphenicol also stalled ribosomes in the cat-86 regulatory leader such that the aminoacyl site was occupied by codon 6, we separately changed leader codons 3, 4, 5, and 6 to the translation termination (ochre) codon TAA. Each of the mutated genes was tested for its ability to be induced by chloramphenicol. The results show that replacement of leader codons 3, 4, or 5 by the ochre codon blocked induction, whereas replacement of leader codon 6 by the ochre codon permitted induction. Collectively, these observations lead to the conclusion that cat-86 induction requires ribosome stalling in leader mRNA, and they identify leader codon 6 as the codon most likely to be occupied by the aminoacyl site of a stalled ribosome that is active in the induction.
Asunto(s)
Acetiltransferasas/genética , Cloranfenicol/farmacología , Ribosomas/efectos de los fármacos , Acetiltransferasas/biosíntesis , Aminoácidos/metabolismo , Bacillus subtilis , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa , Codón , Plásmidos , Procesamiento Proteico-Postraduccional/efectos de los fármacosRESUMEN
The plasmid gene cat-86 is induced by chloramphenicol in Bacillus subtilis, resulting in the synthesis of the gene product chloramphenicol acetyltransferase. Induction is due to a posttranscriptional regulatory mechanism in which the inducer, chloramphenicol, activates translation of cat-86 mRNA. We have suggested that chloramphenicol allows ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the coding sequence. In the present report we show that cat-86 expression can be activated by stalling ribosomes in the act of translating a regulatory leader peptide. Stalling was brought about by starving host cells for specific leader amino acids. Ribosomal stalling, which led to cat-86 expression, occurred upon starvation for the amino acid specified by the leader codon located immediately 5' to the RNA stem-loop structure and was independent of whether that codon specified lysine or tyrosine. These observations support a model for chloramphenicol induction of cat-86 in which the antibiotic stalls ribosome transit in the regulatory leader. Stalling of ribosomes in the leader can therefore lead to destabilization of the RNA stem-loop structure.