Biomedical subjects
Publications and source records attributed to T A Bickle.
The process of DNA recognition by the activated form of the restriction endonuclease from E. coli K involves three enzyme-DNA complexes which can be differentiated experimentally. These are: an initial complex formed at a nonspecific site; a recognition complex involving the host specificity site; and a cleavage complex dependent on the presence of ATP.
The restriction endonuclease from Escherichia coli K specifically cleaves foreign DNA in the presence of S-adenosylmethionine, ATP, and Mg2+. The role of S-adenosylmethionine in this reaction has been studied by following the specific binding of the enzyme to unmodified DNA. The results indicate that S-adenosylmethionine acts as an allosteric effector. However, the rate-limiting step in the activation of the enzyme is not the binding of the effector itself, but an event subsequent to it. The interaction of the S-adenosylmethionine with two mutant K restriction endonucleases isolated previously has also been investigated. One of them, which is defective in restriction, can be activated in a manner similar to the wild type enzyme, while the other one, which lacks both restriction and modification activities (due to a mutation in the subunit responsible for DNA recognition), shows no such effect.
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The 30S ribosomal subunit of E. coli was treated with the bifunctional reagent bis-(methyl)suberimidate. Crosslinked ribosomal proteins were identified as bands with increased molecular weight after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The pattern of crosslinked products was altered when unfolded subunits were used. Free ribosomal protein was not crosslinked. Several of the crosslinked products were cleaved by ammonolysis to form the original monomeric protein constituents. The low yields of the reactions necessitated the use of radioactive proteins and auto-radiographic procedures. The crosslinked proteins were tentatively identified by coelectrophoresis of the radioactive ammonolysis products with carrier 30S protein in sodium dodecyl sulphate, and coelectrophoresis at pH 4.5 in buffers containing urea.
The ribosomal proteins from nine species of prokaryotes have been compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The stained gels were scanned spectrophotometrically, the weight and number average molecular weights were calculated, and the detailed distribution of the proteins as a function of molecular weight was determined. By all of these criteria, the ribosomal proteins from all the species closely resembled each other, despite differences in the pattern of protein bands by conventional disc-gel electrophoresis. Therefore, it is suggested that the structural requirements for the assembly of ribosomal subunits have imposed limitations on the evolution of the ribosomal proteins and that their size has been highly conserved.
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Early attempts to generate new restriction specificities by recombination between allelic restriction-modification systems have been unsuccessful. Bullas et al. succeeded in isolating a new specificity, SQ, in Salmonella that they interpreted as being the result of a recombination event between the parental strains, Salmonella typhimurium and S. postdam, which encode the SB and SP restriction systems, respectively. This interpretation has recently been confirmed by DNA heteroduplex studies with the SB, SP and SQ structural genes. We have determined the DNA sequences recognized by the SB and SP enzymes and found that, like all type I restriction sequences, they are split into two specific domains by a spacer of nonspecific sequence that, for both SB and SP, is 6 base pairs (bp) long. We have now determined the sequence recognized by the recombinant SQ enzyme and find that it is a hybrid between the SB and SP sequences, containing one specific domain from each parental strain. This result implies that each of the two specific domains is recognized by a physically distinct part of the enzyme.