Modulation of the stability of the Salmonella fourU-type RNA thermometer

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values delta G for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements. Physiological Mg2+ concentrations reduce enthalpy and entropy values of uncorrelated base pair opening processes for almost all nucleobases

Expected Basal Insulin Requirement during CSII therapy by Age Group, Sex and BMI, based on 25,718 Young People with Type 1 Diabetes in the DPV Registry.

Background Since the introduction of insulin pumps into the therapy of paediatric subjects, different approaches have been taken to find optimal basal rates. Previously, the DPV registry provided circadian basal rate patterns for different age groups. As the number of pump users has increased recently and short-acting insulin analogues are now predominant, we performed a new analysis with a larger data pool. Methods We included all recent basal profiles from T1D patients between 1 and 25 years from the DPV 2021 data pool. We excluded night-time-only pump users, human regular insulin users, and daily basal rates 1.0 U/kgBW/d. Results In the analysis of profiles from 25,718 young persons with T1D, differences in the daily pattern of basal rates were found between age groups. In addition, we saw significant (p<0.001) differences in total daily basal dose between genders in all age groups except adults. In addition, the shape of the expected basal-rate pattern differed by BMI, HbA1c and use of continuous glucose monitoring. Discussion This analysis demonstrates multiple factors influencing basal patterns and insulin requirement, including age group, gender, overweight, HbA1c, bolus frequency and sensor use. As circadian basal rates are still mandatory for initiating insulin pump therapy with or without automation, a multimodal approach is necessary to estimate optimal basal rates

Evolution from the Prokaryotic to the Higher Plant Chloroplast Signal Recognition Particle: The Signal Recognition Particle RNA Is Conserved in Plastids of a Wide Range of Photosynthetic Organisms

The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplast-specific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system

Modulation of the stability of the Salmonella fourU-type RNA thermometer

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values ΔG for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements

Modulation of the stability of the Salmonella fourU-type RNA thermometer

RNA thermometers are translational control elements that regulate the expression of bacterial heat shock and virulence genes. They fold into complex secondary structures that block translation at low temperatures. A temperature increase releases the ribosome binding site and thus permits translation initiation. In fourU-type RNA thermometers, the AGGA sequence of the SD region is paired with four consecutive uridines. We investigated the melting points of the wild-type and mutant sequences. It was decreased by 5°C when a stabilizing GC basepair was exchanged by an AU pair or increased by 11°C when an internal AG mismatch was converted to a GC pair, respectively. Stabilized or destabilized RNA structures are directly correlated with decreased or increased in vivo gene expression, respectively. Mg2+ also affected the melting point of the fourU thermometer. Variations of the Mg2+ concentration in the physiological range between 1 and 2 mM translated into a 2.8°C shift of the melting point. Thus, Mg2+ binding to the hairpin RNA is regulatory relevant. Applying three different NMR techniques, two Mg2+ binding sites were found in the hairpin structure. One of these binding sites could be identified as outer sphere binding site that is located within the fourU motif. Binding of the two Mg2+ ions exhibits a positive cooperativity with a Hill coefficient of 1.47. Free energy values ΔG for Mg2+ binding determined by NMR are in agreement with data determined from CD measurements

Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution

In prokaryotes, RNA thermometers regulate a number of heat shock and virulence genes. These temperature sensitive RNA elements are usually located in the 5′-untranslated regions of the regulated genes. They repress translation initiation by base pairing to the Shine–Dalgarno sequence at low temperatures. We investigated the thermodynamic stability of the temperature labile hairpin 2 of the Salmonella fourU RNA thermometer over a broad temperature range and determined free energy, enthalpy and entropy values for the base-pair opening of individual nucleobases by measuring the temperature dependence of the imino proton exchange rates via NMR spectroscopy. Exchange rates were analyzed for the wild-type (wt) RNA and the A8C mutant. The wt RNA was found to be stabilized by the extraordinarily stable G14–C25 base pair. The mismatch base pair in the wt RNA thermometer (A8–G31) is responsible for the smaller cooperativity of the unfolding transition in the wt RNA. Enthalpy and entropy values for the base-pair opening events exhibit linear correlation for both RNAs. The slopes of these correlations coincide with the melting points of the RNAs determined by CD spectroscopy. RNA unfolding occurs at a temperature where all nucleobases have equal thermodynamic stabilities. Our results are in agreement with a consecutive zipper-type unfolding mechanism in which the stacking interaction is responsible for the observed cooperativity. Furthermore, remote effects of the A8C mutation affecting the stability of nucleobase G14 could be identified. According to our analysis we deduce that this effect is most probably transduced via the hydration shell of the RNA