Genetic Screen of the B. subtilis pbuE Adenine-Responsive Riboswitch Expression Platform Reveals Preferences for Base Pairing in the Nucleator Hairpin-Stem

Located on the 5&rsquo; untranslated regions of bacterial messenger RNA, riboswitches are regulatory structures that are responsible for the modulation of genetic expression through ligand-dependent binding. Inclusive of two components, the riboswitch will probe its environment without the aid of an additional protein or DNA structure to sense and attach a specific metabolite to the region known as the upstream aptamer domain. The downstream expression platform then endures changes in its folding pattern to adopt one of two secondary structures, resulting in either the inhibition or continuation of mRNA production. Due to its smaller size, the B. subtilis pbuE adenine-responsive riboswitch has been the focus of many previous studies that sought to determine how the tertiary structure of the aptamer domain allows for tight binding with high specificity. The expression platform, however, is similarly interesting, as it participates in strand invasion in order to produce a transcription terminating hairpin that is rho independent. Through the mutagenic cloning of a novel pbuE variant named NH5, the investigation into the reduced nucleator Hairpin-Stem library containing 6 randomized nucleotides revealed a strict preference for genetic base pairing proximal to the L4 loop. Additionally, the data suggests that weak A-U and G-U interactions or even non-canonical coupling between the nucleobases furthest from the polyuridine tract is tolerated if supplemented with three strong Watson-Crick pairs. Paving the way for the creation of additional synthetic riboswitch structures, the robust screening of the P4 region allows for a more thorough understanding of the fundamental requirements that promote the formation of the terminator helix and the subsequent mechanism of strand invasion.</p

The metabolism of Brucellae: The nature of the effects of pH and concentration on the rate of oxidation of succinate

The sharp rise in the rate of oxidation of succinate by Brucella abortus that occurs with gross increases in hydrogen ion or the substrate concentration was accompanied by an even greater degree of increase in the rate of substrate uptake. The degree of change in these rates and of pyruvate formation with glutamate, a reported precursor of succinate, was minimal in comparison to that with succinate. The optimal pH for oxidation of succinate became strikingly higher with increased concentration of substrate; similarly, concentration optima were observed and they rose with increased pH. An analysis of these data indicated that the activity was largely but not exclusively dependent on the concentration of undissociated molecules. Contrary to these findings with intact cells, crude succinoxidase preparations exhibited a constant pH optimum at neutrality with changes in substrate concentration. The results favored a conclusion that permeability is rate limiting in the oxidation of succinate by the organism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32510/1/0000600.pd