Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth.
The groES and groEL genes of Escherichia coli have been shown previously to belong to a single operon under heat shock regulation. Both proteins have been universally conserved in nature, as judged by the presence of similar proteins throughout evolution. The GroEL protein has been shown to bind promiscuously to many unfolded proteins, thus preventing their aggregation. ATP hydrolysis by GroEL results in the release of the bound polypeptides, a process that often requires the action of GroES. In an effort to understand GroEL and GroES structure and function, we have determined the nucleotide changes of nine mutant alleles of groEL. All of these mutant alleles were isolated because they block bacteriophage lambda growth. Our sequencing results demonstrate that (i) many of these alleles are identical, in spite of the fact that they were independently isolated, and (ii) most of the different alleles are clustered in the same region of the gene. One of the mutant alleles was shown to possess two nucleotide alterations in the groEL coding phase, one of which is located in a putative ATP-binding domain. The two nucleotide changes were separated by genetic engineering, and each individual change was shown to exert an effect on bacteriophage growth. But, using genetic analyses, we demonstrate that the restriction on bacterial growth at elevated temperatures is conferred only by the mutation within the putative ATP-binding domain. We have cloned the mutant alleles on multicopy plasmids and overexpressed their products. By testing for the ability of bacteriophage either to propagate or to form colonies at 43 degrees C, we have been able to divide the mutant proteins into those with no activity and those with residual activity under the various conditions tested.
Zeilstra-Ryalls
J
J
Department of Cellular, Viral, and Molecular Biology University of Utah, School of Medicine, Salt Lake City.
Fayet
O
O
Baird
L
L
Georgopoulos
C
C
D001426:Bacterial Proteins / Q000235:genetics*; D010582:Bacteriophage lambda / Q000254:growth & development*; D001483:Base Sequence; D018834:Chaperonin 60; D018833:Chaperonins; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006360:Heat-Shock Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D010641:Phenotype; D011506:Proteins / Q000502:physiology; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000254:growth & development*; D013696:Temperature; D014779:Virus Replication
D001426:Bacterial Proteins; D018834:Chaperonin 60; D006360:Heat-Shock Proteins; D011506:Proteins; D018833:Chaperonins