RESUMEN
A mutation in the infA gene encoding initiation factor 1 (IF1) gives rise to a cold-sensitive phenotype. An Escherichia coli strain with this mutation was used as a tool to select for second-site suppressors that compensate for the cold sensitivity and map specifically to rRNA. Several suppressor mutants with altered 16S rRNA that partially restore growth of an IF1 mutant strain in the cold were isolated and characterized. Suppressor mutations were found in helix (h)18, h32, h34 and h41 in 16S rRNA. These mutations are not clustered to any particular region in 16S rRNA and none overlap previously reported sites of interaction with IF1. While the isolated suppressors are structurally diverse, they are functionally related because all affect ribosomal subunit association in vivo. Furthermore, in vitro subunit-association experiments indicate that most of the suppressor mutations directly influence ribosomal subunit association even though none of these are confined to any of the known intersubunit bridges. These results are consistent with the model that IF1 is an rRNA chaperone that induces large-scale conformational changes in the small ribosomal subunit, and as a consequence modulates initiation of translation by affecting subunit association.
Asunto(s)
Frío/efectos adversos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Mutación , Factor 1 Procariótico de Iniciación/metabolismo , ARN Ribosómico 16S/metabolismo , Subunidades Ribosómicas/metabolismo , Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Conformación de Ácido Nucleico , Factor 1 Procariótico de Iniciación/genética , Multimerización de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/química , ARN Ribosómico 16S/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Supresión GenéticaRESUMEN
Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli. The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted. Here, we describe a set of suppressor mutations that are located in the processing stem of precursor 23S rRNA. These mutations interfere with processing of the 23S rRNA termini. A lesion of RNase III also suppresses the cold sensitivity. Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially compensate for this defect by disrupting rRNA maturation. These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation.