RESUMEN
The StrR protein is a DNA-binding protein activating the transcription of streptomycin biosynthesis of Streptomyces griseus N2-3-11 and Streptomyces glaucescens. A putative helix-turn-helix motif located between amino acid positions 207 and 227 of the StrR protein was identified as a prerequisite for its DNA-binding properties. Although, C-terminal truncated StrR proteins were able to interact with StrR-binding sites, they failed to activate transcription from the StrR-dependent promotor strB1p. Therefore, the C-terminal domain of StrR seemed to be necessary for its function as transcriptional activator.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Streptomyces griseus/metabolismo , Estreptomicina/biosíntesis , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Secuencias Hélice-Asa-Hélice/genética , Datos de Secuencia Molecular , Mutagénesis/fisiología , Plásmidos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli. The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA. In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA. The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice. These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible. The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models.
Asunto(s)
Escherichia coli/ultraestructura , Procesamiento de Imagen Asistido por Computador , ARN Bacteriano/ultraestructura , ARN Ribosómico/ultraestructura , Secuencia de Bases , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
A DNA fragment containing the Escherichia coli 5S rDNA sequence linked to a T7 promoter was prepared by PCR from an M13 clone carrying the 5S-complementary sequence. The DNA was transcribed with T7 polymerase using a mixture of [alpha-32P]UTP and 4-thio-UTP, yielding a transcript in which approximately 18% of the uridine residues were randomly replaced by thiouridine. This modified 5S RNA could be reconstituted efficiently into 50S ribosomal subunits or 70S functional complexes. The reconstituted particles were irradiated at wavelengths above 300 nm, and the crosslinked ribosomal components were identified. A crosslink in high yield was reproducibly observed between the modified 5S RNA and 23S RNA, involving residue U-89 of the 5S RNA (at the loop end of helix IV) linked to nucleotide 2477 of the 23S RNA in the loop end of helix 89, immediately adjacent to the peptidyltransferase "ring." On the basis of this result, and in combination with earlier immunoelectron microscopic data, we propose a model for the orientation of the 5S RNA in the 50S subunit.
Asunto(s)
Conformación de Ácido Nucleico , Peptidil Transferasas/metabolismo , ARN Ribosómico 5S/química , ARN Ribosómico 5S/metabolismo , Secuencia de Bases , Cartilla de ADN , ADN Ribosómico/química , ADN Ribosómico/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Modelos Estructurales , Datos de Secuencia Molecular , Peptidil Transferasas/biosíntesis , Peptidil Transferasas/química , Reacción en Cadena de la Polimerasa , ARN Ribosómico 5S/biosíntesis , ADN Polimerasa Dirigida por ARN , Ribonucleasa H , Ribosomas/metabolismo , Transcripción Genética , Uridina Trifosfato/metabolismoRESUMEN
Using two synthetic deoxyribonucleotide probes encoding segments of the primary structure of initiation factor IF3 from Bacillus stearothermophilus, we identified and cloned a segment of DNA which carries the infC gene. As in Escherichia coli, the infC gene begins with the unusual initiation triplet AUU, and is followed by the structural genes for ribosomal proteins L35 and L20 (rpmI and rplT, respectively).
Asunto(s)
Proteínas Bacterianas/genética , Genes Bacterianos , Geobacillus stearothermophilus/genética , Familia de Multigenes , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Genes , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Mapeo RestrictivoRESUMEN
A 7 kb chromosomal DNA fragment from R. melilotii was cloned, which complemented temperature-sensitivity of an E. coli amber mutant in rpsA, the gene for ribosomal protein S1 (ES1). From complementation and maxicell analysis a 58 kd protein was identified as the homolog of protein S1 (RS1). DNA sequence analysis of the R. melilotii rpsA gene identified a protein of 568 amino acids, which showed 47% identical amino acid homology to protein S1 from E. coli. The RS1 protein lacked the two Cys residues which had been reported to play an important role for the function of ES1. Two repeats containing Shine-Dalgarno sequences were identified upstream of the structural gene. Binding studies with RNA polymerase from E. coli and Pseudomonas putida located one RNA-polymerase binding site close to the RS1 gene and another one several hundred basepairs upstream. One possible promoter was also identified by DNA sequence comparison with the corresponding E. coli promoter.
Asunto(s)
Proteínas Bacterianas/genética , Clonación Molecular , Genes Bacterianos , Rhizobium/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Fosfoproteínas/metabolismo , PlásmidosRESUMEN
The genes for the ribosomal proteins S9 (rpsI) and L13 (rplM) of Escherichia coli have been cloned into a lambda phage vector termed L47.1. The two genes were identified by infecting UV-light irradiated cells with the resultant phages and analyzing the protein products by two-dimensional gel electrophoresis. Suitable DNA fragments of the isolate were cloned subsequently into M13 phage vectors and their nucleotide sequence was determined by the dideoxy method. It is evident that the two genes form a transcriptional unit, the rplM gene being promoter-proximal. There is a typical signal sequence for transcriptional termination after the rpsI gene. The codon usage pattern in the two genes is similar to other ribosomal protein genes of E. coli.