Analysis of the proteins synthesized in ultraviolet light-irradiated Escherichia coli following infection with the bacteriophages λ drif d 18 and λ dfus -3

The presence of EF-Tu, RNA polymerase subunit α, and EF-G on the λ dfus -3 genome and EF-Tu, ribosomal proteins L7/L12, and RNA polymerase subunit β on the λ drif d 18 genome has been confirmed using a two-dimensional gel electrophoresis technique sensitive to changes in isoelectric point and molecular weight. In this system two EF-Tu gene products could not be resolved. Following infection of ultraviolet light-irradiated Escherichia coli with either λ dfus -3 or λ drif d 18, the EF-Tu gene, tufA , near 65 minutes on the genetic map is expressed as 3–4 copies per EF-G molecule. The EF-Tu gene, tufB , near 79 minutes on the genetic map, is expressed at about one-third of this rate. α is expressed as 1 copy per EF-G molecule, β as 0.14 per EF-G molecule and L7/L12 as 2.5 per EF-G. These figures compare well with the relative amounts found in exponentially-growing cells, in which the ratio of EF-Tu to EF-G is approximately 5. Almost 90% of the total number of proteins (calculated on a molecular weight basis) which theoretically can be encoded on the λ drif d 18 have been identified on the two-dimensional gel.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47541/1/438_2004_Article_BF00341733.pd

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu

The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86 , 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47543/1/438_2004_Article_BF00401747.pd