RESUMEN
We describe the crystallization and structure determination of the 30 S ribosomal subunit from Thermus thermophilus. Previous reports of crystals that diffracted to 10 A resolution were used as a starting point to improve the quality of the diffraction. Eventually, ideas such as the addition of substrates or factors to eliminate conformational heterogeneity proved less important than attention to detail in yielding crystals that diffracted beyond 3 A resolution. Despite improvements in technology and methodology in the last decade, the structure determination of the 30 S subunit presented some very challenging technical problems because of the size of the asymmetric unit, crystal variability and sensitivity to radiation damage. Some steps that were useful for determination of the atomic structure were: the use of anomalous scattering from the LIII edges of osmium and lutetium to obtain the necessary phasing signal; the use of tunable, third-generation synchrotron sources to obtain data of reasonable quality at high resolution; collection of derivative data precisely about a mirror plane to preserve small anomalous differences between Bijvoet mates despite extensive radiation damage and multi-crystal scaling; the pre-screening of crystals to ensure quality, isomorphism and the efficient use of scarce third-generation synchrotron time; pre-incubation of crystals in cobalt hexaammine to ensure isomorphism with other derivatives; and finally, the placement of proteins whose structures had been previously solved in isolation, in conjunction with biochemical data on protein-RNA interactions, to map out the architecture of the 30 S subunit prior to the construction of a detailed atomic-resolution model.
Asunto(s)
Ribosomas/química , Thermus thermophilus/química , Cristalización , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Lutecio/metabolismo , Modelos Moleculares , Peso Molecular , Osmio/metabolismo , Conformación Proteica , Subunidades de Proteína , Ribosomas/metabolismo , SolventesRESUMEN
Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.
Asunto(s)
Factor 1 Eucariótico de Iniciación/química , ARN Ribosómico 16S/química , Proteínas Ribosómicas/química , Ribosomas/química , Thermus thermophilus/química , Emparejamiento Base , Sitios de Unión , Cristalografía por Rayos X , Factor 1 Eucariótico de Iniciación/metabolismo , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , Estructura Secundaria de Proteína , ARN Ribosómico 16S/metabolismo , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoAsunto(s)
Antibacterianos/química , Conformación de Ácido Nucleico , ARN Ribosómico/química , Proteínas Ribosómicas/química , Secuencia de Bases , Sitios de Unión , Ligandos , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , ARN Bacteriano/química , ARN Ribosómico 16S/química , Thermus thermophilus/genéticaRESUMEN
Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.
Asunto(s)
ARN Ribosómico/química , Proteínas Ribosómicas/química , Ribosomas/química , Proteínas Bacterianas/química , Cristalografía por Rayos X , Sustancias Macromoleculares , Modelos Moleculares , Conformación de Ácido Nucleico , Conformación Proteica , ARN Bacteriano/química , Thermus thermophilusRESUMEN
The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.
Asunto(s)
Antibacterianos/química , Ribosomas/química , Antibacterianos/farmacología , Sitios de Unión , Cristalografía por Rayos X , Código Genético , Sustancias Macromoleculares , Modelos Moleculares , Imitación Molecular , Conformación de Ácido Nucleico , Paromomicina/química , Paromomicina/farmacología , Conformación Proteica , ARN Bacteriano/química , ARN Bacteriano/fisiología , ARN Mensajero/metabolismo , ARN Ribosómico/química , ARN Ribosómico/fisiología , ARN Ribosómico 16S/química , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/fisiología , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Espectinomicina/química , Espectinomicina/farmacología , Estreptomicina/química , Estreptomicina/farmacología , Relación Estructura-Actividad , Thermus thermophilusRESUMEN
We have used the recently determined atomic structure of the 30S ribosomal subunit to determine the structures of its complexes with the antibiotics tetracycline, pactamycin, and hygromycin B. The antibiotics bind to discrete sites on the 30S subunit in a manner consistent with much but not all biochemical data. For each of these antibiotics, interactions with the 30S subunit suggest a mechanism for its effects on ribosome function.