Allosteric regulation is a fundamental principle in nature. Most enzymes working in metabolic pathways are regulated by interaction with other molecules acting as allosteric cofactors. Over the past decade, RNA conformation has been shown to respond to external stimuli. A number of ligand-specific molecular switches that are composed of aptamers attached to ribozyme structures have been developed.[1] Binding of a specific ligand to the aptamer domain either stabilizes or destabilizes the active conformation of the catalytic domain and results in an increase or decrease in catalytic activity. Riboswitches have also been discovered in nature; the mRNA conformation is changed upon interaction with a specific ligand and this results in inhibition/disruption of transcription or translation.[2] Although natural riboswitches can run through many cycles of switching, artificial riboswitches do not work in a reversible mode: the activity is either switched on or off in response to the addition of the allosteric cofactor. We set out to develop a system that has the potential to be regulated in a reversible manner. To this end, we have designed a hairpin ribozyme variant whose catalytic properties are dependent on flavine mononucleotide (FMN). Herein we show that ribozyme activity is switched on in the presence of FMN in the oxidized state, whereas under reducing conditions, FMN is released from its binding site and a clear decline in activity results.To construct hairpin ribozyme variants that can be regulated in an allosteric way, we decided to replace helix 4 of the wild-type hairpin ribozyme [3] (Figure 1) with a specific sequence. This sequence acts as a communication module and connects the ribozyme moiety with the FMN-specific aptamer, which was previously identified by in vitro selection. [4] The 11-nucleotide bulge segment (nucleotides 8-13 and 24-28 in Figure 1 b) has been used before for the construction of FMN-dependent hammerhead aptazymes [5] and for competitive regulation of modular allosteric aptazymes by a small molecule and an oligonucleotide effector.[6] Furthermore, a natural RNA fold within mRNA has been discovered that specifically interacts with FMN to regulate the expression of bacterial genes involved in the biosynthesis and transport of riboflavin and FMN. [7] We have engineered two variants of hairpin aptazymes, HPAR2 and HPAR5 (Figure 1). In the absence of FMN, we expected the conformation of loop B to be less stable. Binding of FMN to the aptamer domain should trigger a conformational change within the bridge that in turn should cause a structural rearrangement of the adjoining ribozyme, thus dictating its activity. The communication module used in HPAR2 was developed by in vitro selection

Nimmerichter, A, Novak, N, Triska, C, Prinz, B, and Breese, BC. Validity of treadmill-derived critical speed on predicting 5,000-meter track-running performance. J Strength Cond Res 31(3): 706-714, 2017-To evaluate 3 models of critical speed (CS) for the prediction of 5,000-m running performance, 16 trained athletes completed an incremental test on a treadmill to determine maximal aerobic speed (MAS) and 3 randomly ordered runs to exhaustion at the [INCREMENT]70% intensity, at 110% and 98% of MAS. Critical speed and the distance covered above CS (D') were calculated using the hyperbolic speed-time (HYP), the linear distance-time (LIN), and the linear speed inverse-time model (INV). Five thousand meter performance was determined on a 400-m running track. Individual predictions of 5,000-m running time (t = [5,000-D']/CS) and speed (s = D'/t + CS) were calculated across the 3 models in addition to multiple regression analyses. Prediction accuracy was assessed with the standard error of estimate (SEE) from linear regression analysis and the mean difference expressed in units of measurement and coefficient of variation (%). Five thousand meter running performance (speed: 4.29 ± 0.39 m·s; time: 1,176 ± 117 seconds) was significantly better than the predictions from all 3 models (p < 0.0001). The mean difference was 65-105 seconds (5.7-9.4%) for time and -0.22 to -0.34 m·s (-5.0 to -7.5%) for speed. Predictions from multiple regression analyses with CS and D' as predictor variables were not significantly different from actual running performance (-1.0 to 1.1%). The SEE across all models and predictions was approximately 65 seconds or 0.20 m·s and is therefore considered as moderate. The results of this study have shown the importance of aerobic and anaerobic energy system contribution to predict 5,000-m running performance. Using estimates of CS and D' is valuable for predicting performance over race distances of 5,000 m.

These findings support previous laboratory-based studies demonstrating reductions in GE with increasing cadence and gradient that might be attributed to changes in muscle-activity pattern.

No abstract

Allosterische Regulation ist ein fundamentales Prinzip in der Natur. Die meisten Enzyme metabolischer Pfade werden durch Interaktionen mit anderen Molekülen reguliert, die als allosterische Cofaktoren wirken. Im Laufe der letzten zehn Jahre hat sich bestätigt, dass auch die Konformation von RNA-Molekülen extern gesteuert werden kann. Durch Verbindung von Ribozymen mit spezifischen Aptameren wurde eine Reihe Liganden-spezifischer molekularer Schalter entwickelt.[