Chemical probing methods have been used to "footprint" 16S ribosomal RNA (rRNA) at each step during the in vitro assembly of twenty 30S subunit ribosomal proteins. These experiments yield information about the location of each protein relative to the structure of 16S rRNA and provide the basis for derivation of a detailed model for the three-dimensional folding of 16S rRNA. Several lines of evidence suggest that protein-dependent conformational changes in 16S rRNA play an important part in the cooperativity of ribosome assembly and in fine-tuning of the conformation and dynamics of 16S rRNA in the 30S subunit.

Escherichia coli thymidylate synthase (TS) is a dimeric protein containing identical subunits. When R126E, an inactive mutant of this enzyme, was incubated at room temperature with other inactive mutants of E. coli, TS enzyme activity gradually reappeared. The rate of activity restoration was dependent on the mutant employed. In the case of C146W, an active site mutant, the half-time required for maximal activity restoration was about one hour, which was about 500-fold faster than that obtained with C146S. The final specific activity of the mutant mixtures, based on the concentration of R126E, was equivalent to that of the wild-type TS (WT-TS). However, when the activities of E. coli WT-TS and mutant TS mixtures were compared for their extents of renaturation following denaturation as described for Lactobacillus casei TS [Pookanjanatavip, M., Yuthavong, Y., Greene, P. J., & Santi, D. V. (1992) Biochemistry 31, 10303-10309], only about one-half of the activity of WT-TS was restored, implying that the denaturation-renaturation procedure was less efficient than allowing the native TS mutant dimers to exchange subunits. If, as proposed, subunit exchange is responsible for the observed restoration of activity to the E. coli mutant TS mixtures, it would suggest that only one active site cysteine, that provided by R126E in the dimer (R126E)-(C146W), is sufficient to yield the same kcat as WT-TS, which contains one active site cysteine in each subunit. Other mutant dimers that contain both active site cysteines such as (R126E)-(Y94A) and (R126E)-(I264Am) are also fully active, even though one of the subunits is functionally inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract

No abstract

In a previous study we demonstrated that Escherichia coli thymidylate synthase activity could be restored completely by incubating basically inactive mutants of this enzyme at room temperature with R(126)E, another inactive mutant [Maley, F., Pedersen-Lane, J., and Changchien, L.-M. (1995) Biochemistry 34, 1469-1474]. Since only one of the enzyme's two subunits possessed a functional active site and the restoration of activity could be titrated to be equivalent to that of the wild-type enzyme's specific activity, it was proposed that thymidylate synthase was a half-of-the-sites activity enzyme. We now provide additional support for this thesis by presenting an in-depth analysis of some conditions affecting the restoration of enzyme activity. For this purpose, we employed two mutants with marginal thymidylate synthase activity, Y(94)A and R(126)E. The parameters that were examined included pH, concentration of protein, temperature, and urea concentration, all of which influenced the rate of activity restoration. It was found, surprisingly, that by maintaining the amount of each protein constant, while increasing the volume of solution, the rate and total activity restored was greatly enhanced. Increasing the pH from 6.0 to 9.0 markedly increased the rate at which the optimal activity was restored, as did increasing the temperature from 4 to 40 degrees C. A similar effect was obtained when the incubation of the mutants was conducted at 4 degrees C in the presence of 1.5 M urea, a temperature at which activity is restored extremely slowly. Raising the pH to 9.0 resulted in an almost instantaneous restoration of activity at 4 degrees C. The manner in which thymidylate synthase activity is restored from the mutants in the presence of varying concentrations of ethanol, ethylene glycol, and glycerol suggests that changes in subunit interaction and enzyme conformation are in part responsible for the observed differences. Most significantly, at solution levels of 10%, ethanol was found to activate, while ethylene glycol inhibited slightly and glycerol was somewhat more inhibitory. At a concentration of 20%, ethanol inhibited rather strikingly, ethylene glycol was slightly more inhibitory than at 10%, and glycerol was strongly inhibitory. Since the net result of these findings is the suggestion that the restoration of thymidylate synthase activity is due to a separation of the mutant dimers into their respective subunits, followed by their recombination to an active heterodimer, evidence for this phenomenon was sought by separating the recombined dimers using nondenaturating polyacrylamide gel electrophoresis. Sequence analysis of the isolated homo- and heterodimers clearly demonstrated that the active enzyme is a product of subunit exchange, one that is very efficient relative to the wild-type enzyme, which did not exchange subunits unless denatured.