Hiroko Nashimoto

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations

SummaryA 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.

Late steps in the assembly of 30 S ribosomal proteins in vivo in a spectinomycin-resistant mutant of Escherichia coli☆

Abstract The late steps in ribosome assembly in vivo were studied by characterizing mutations which suppress the cold-sensitivity of a spectinomycin-resistant mutant of Escherichia coli . The results obtained indicated that the cold-sensitivity could be relieved by secondary alterations in either the S2, S3 or S5 protein of the 30 S ribosomal subunit. The gene controlling the alteration of S2 protein was closely linked to the polC gene located at about 3.5 minutes on the genetic map of E. coli , whereas S3 and S5 suppressor genes were linked to the str-spc region at 64 minutes. A possible model in which the S2, S3 and S5 proteins constitute a sub-assembly pathway in the assembly of 30 S subunits in vivo is discussed.

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12.

SummaryTemperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.

Temperature-sensitive mutations in the α subunit gene of Escherichia coli RNA polymerase☆

Abstract Two temperature-sensitive mutations in the α subunit gene of Escherichia coli RNA polymerase ( rpoA ) were isolated by localized mutagenesis of the aroE - strA region of the chromosome, selecting for strains defective in RNA synthesis at non-permissive temperature. These mutations were mapped within a narrow segment assigned to rpoA , rpsD (S4) and rpsK (S11) genes. RNA polymerase purified from one of the mutants ( ts 112) was more thermolabile than the wild-type enzyme, but after a cycle of reversible dissociation and re-assembly, the thermolabile enzyme became even more thermostable than the wild-type enzyme. On the other hand, RNA polymerase purified from another mutant ( ts 101) was more thermostable to start with than the wild-type enzyme. By reversible dissociation and cross-assembly of these purified enzymes, mutational alterations were demonstrated to have occurred within the α subunit of the enzyme for both of the mutant strains. Transcriptional fidelity of these mutant RNA polymerases was greatly impaired, and frequent misincorporations of adenine and uracil in place of guanine and cytosine were noted in in vitro reactions at 30 °C and 42 °C. Nevertheless, the mutant strains showed no indication of erroneous transcription in vivo .

Cloning of an EcoRI fragment carrying E. coli tufA gene

SummaryEcoRI fragments of the transducing phage λfus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Apr) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and its EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA·DNA hybridization of RNA transcribed from phage λrifd18 carrying tufB with DNA from pTUAl.

Indole as an activator for in vitro attachment of tail fibers in the assembly of bacteriophage T4D

Abstract The in vitro attachment of the completed tail-fibers to otherwise completed fiberless phage particle is activated by a thermostable, dialyzable cofactor, which can effectively be replaced by a low concentration of indole. Kinetic evidence suggests that the cofactor acts as an allosteric effector, which activates the fiberless particle to act as receptor for the tail fibers.