Thermodynamics and Kinetics of Conformational Transitions in Oligonucleotides and tRNA

Kinetics of duplex formation for individual DNA strands within a single protein nanopore

“…The value for the activation enthalpy is in line with literature values for duplex formation in solution (34,40), and the value for the activation entropy is 40% lower than values for similar dissociation events in solution (41).…”

Formation of metastable RNA structures by sequential folding during transcription: Time-resolved structural analysis of potato spindle tuber viroid (−)-stranded RNA by temperature-gradient gel electrophoresis

“…TGGE of transcript PSTVd-Sty 1 under equilibrium conditions+ The transcript in 0+2ϫ TBE was denatured at 70 8C for 15 min and slowly renatured to room temperature with ;0+1 8C/min+ PAGE: 4% PA 30:1, 0+2ϫ TBE, silver-stained+ The marker lanes contain a crude RNA extract with 7: 7 S RNA; P: PSTVd; l: linear PSTVd; c: circular PSTVd+ Circular and linear PSTVd comigrate at low temperature because their shape is nearly identical+ The completely denatured molecules migrate very differently because of the high retardation of the covalently closed circle+ possible through a variety of parameters (temperature, anion concentration, viscosity, specific inhibitors) that, however, also influence RNA structure formation+ Therefore, we have chosen to vary the concentration of NTP, which is known to regulate the transcription rate in a wide range (Masukata & Tomizawa, 1990;Chamberlin & Ring, 1973) without altering the RNA structure+ All other conditions (see Materials & Methods) were kept constant+ For all experiments, the polymerase is used in a 24ϫ molar excess over the template+ Lowering the transcription rate does not lead to an increase in non-full-length transcripts (see Fig+ 3)+ Thus all different bands visible in TGGE will consist of structures with the same sequence and length, differing only in shape+ Of course, prematurely terminated transcripts are also generated, but their size heterogeneity will prohibit their detection in gels+ Furthermore, the absolute yields of transcript are lowered with decreasing concentrations of NTP, but the total amount of NTPs is not limiting for the yields; after 15 min of transcription the NTP concentration is lowered by less than 5% with all NTP concentrations used+ For determination of transcription rates, the amount of transcripts was determined at 15 min (see Fig+ 4); at that time point the rates were influenced neither by a lag phase at short times nor by saturation effects at long times (data not shown)+ At NTP concentrations below 50 mM a sigmoidal dependence of yields is obvious; this is a well known effect (Chamberlin & Ring, 1973;Kadesch & Chamberlin, 1982)+ We have no explanation for the step-like dependence near 300 mM NTP+ From the concentration dependence of the transcript yields, however, values for the transcription rates can be derived+ The value of 230 6 20 nt/s at c NTP ϭ 400 mM and 37 8C is reduced to ;50 nt/s at 20 8C (Chamberlin & Ring, 1973)+ Using that value as a basis of calculation, the rates determined here are roughly between 3 nt/s at c NTP ϭ 30 mM and 20 nt/s at c NTP ϭ 70 mM; that is, we are able to shift the transcription rate of the T7-polymerase into the proper range for polymerase II+ According to these rates of transcription, synthesis of a full-length PSTVd transcript needs between a few seconds and several minutes+ Because folding of RNA structures (for review see Turner et al+, 1990;Riesner & Römer, 1973) requires between microseconds (elongation of helices) and minutes (closure of complex junctions or tertiary foldings), the process of transcription and the process of folding might interfere with each other+…”

“…NTP denotes an unlimited supply for production of transcripts+ The polymerase synthesizes partial transcripts p that fold into different structures depending on their length: (1) If synthesis is fast only small hairpins are formed and the resulting full-length transcript F meta is also highly metastable+ (2) If synthesis is slow the partial transcript p has sufficient time to rearrange into p r , for example by formation of junctions, and the resulting full-length transcripts F r,meta have structures of higher stability+ These consequences follow quite easily from the known rates of synthesis and RNA structure formation (for review see Turner et al+, 1990;Riesner & Römer, 1973), respectively+ For example, the relaxation times for formation of small hairpin loops at room temperature are in the microsecond range, whereas relaxation times of clover leaf formation are in the range of seconds+ After synthesis the various metastable structures (F meta and F r,meta ) can rearrange into structures of higher thermodynamic stability+ As an example, F meta might rearrange into the thermodynamic optimum S opt but also into a structure S inter of intermediate stability; the metastable structure F r,meta can also rearrange into the optimal structure S opt but with a higher activation energy+ A rearrangement of F r,meta into a structure similar to S inter should also be possible, but is not introduced here into the reaction scheme because of the higher stability of F r,meta in comparison to F meta + Resulting concentrations for the various structures as calculated by KinSim (Barshop et al+, 1983;Dang & Frieden, 1997;KinSim, 1998) are shown in Figure 11+ The rate constants (in arbitrary units) used for calculations are (1) k 1 ϭ 1+0 or 0+08, the first implying fast and the second slow transcription+ (2) k 4 ϭ 1+0, which is at least as fast as the synthesis steps resulting in a high concentration of transcripts S opt with optimal structures and very low concentrations of their metastable precursors F meta + (3) k 7 ϭ 0+5 allows for formation of a quite stable but nevertheless metastable structure S inter in parallel to formation of the optimal structure S opt out of the metastable precursors F meta + (4) k 5 ϭ 0+1, which is slower than k 4 , resulting in accumulation of the metastable precursor F r,meta ; that is, rearrangement from F r,meta into the opti-mal structures S opt is kinetically blocked in comparison to the rearrangement of F meta into S opt + (5) k 6 ϭ 0+5 gives rise to the two major parallel pathways; its value in relation to that of the synthesis steps (k 1 ) determines the relative population of both pathways+ Figure 11A might be compared qualitatively with Figure 7+ Obviously the relative concentrations of structures in dependence upon transcription rate are modeled adequately; that is, the optimal structure S opt changes only slightly in relative concentration when the transcription rate is changed by orders of magnitude; the relative concentration of metastable structures P/Q and S inter increases more than that of the optimal structure; and the relative concentration of the metastable structure N and F r,meta , respectively, drops significantly+ Next we compare Figure 11B with Figure 9A+ The time dependence of the experimentally determine...…”

Thermostability of double-stranded deoxyribonucleic acids: the effects of covalent additions of a psoralen

“…Thus, the melting curves were measured at essentially equilibrium conditions, since the relaxation times of the double-stranded vs. single-stranded oligonucleotide equilibria at our conditions are much smaller than 1 s as estimated on the basis of kinetic data of oligonucleotides (Riesner & Romer, 1973;Craig et al, 1971;Porschke & Eigen, 1971).…”