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T C Gluick

Publications and source records attributed to T C Gluick.

7 recordsLinked to original sources
PubMed1997

Folding of an mRNA pseudoknot required for stop codon readthrough: effects of mono- and divalent ions on stability.

Unfolding of an mRNA pseudoknot that induces ribosome suppression of the gag gene stop codon in Moloney murine leukemia virus has been studied by UV hyperchromicity and calorimetry. The pseudoknot melts in two steps, corresponding to its two helical stems. The total enthalpy of denaturation is approximately 170 kcal/mol, approximately the value expected for the secondary structure. At low salt concentrations (<50 mM KCl) the unfolding transitions are not two-state, but they approach two-state behavior at higher salt concentrations. The structure is preferentially stabilized by smaller alkali metal ions (Li+ > Na+ > K+ > Rb+ > Cs+) and by NH4+; the same preferences are exhibited by one of the stems in the context of a hairpin. Divalent metal ions are not required to fold the pseudoknot but do stabilize it further. To examine divalent ion effects over a wide concentration range, urea was used to lower the RNA unfolding temperature and was shown not to affect characteristics of the pseudoknot unfolding in other respects. The pseudoknot binds divalent ions somewhat more tightly than a hairpin but shows only weak selectivity for different size ions. It is suggested that a region of "intermediate" divalent ion binding affinity, in between highly ligated specific sites and purely delocalized ion binding in character, is created by the pseudoknot fold but that nonspecific, delocalized ion binding contributes at least half the free energy of pseudoknot stabilization by Mg2+.

Animals↗
PubMed1997

Effects of Mg2+, K+, and H+ on an equilibrium between alternative conformations of an RNA pseudoknot.

A complex pseudoknot structure surrounds the first ribosome initiation site in the Escherichia coli alpha mRNA and mediates its regulation by ribosomal protein S4. A 112 nt RNA fragment containing this pseudoknot exists in two conformations that are resolvable by gel electrophoresis below room temperature. Between 30 degrees C and 45 degrees C the conformers reach thermodynamic equilibrium on a time scale ranging from one hour to one minute, and the interconversion between conformers is linked to H+, K+ and Mg2+ concentrations. Mg2+ favors formation of the "fast" electrophoretic form: a single Mg2+ is bound in the rate-limiting step, followed by cooperative binding of approximately 1.7 additional ions. Binding of the latter ions provides most of the favorable free energy for the reaction. However, the "slow" form binds about the same number of Mg ions, albeit more weakly, so that saturating Mg2+ concentrations drive the equilibrium to only approximatley 70% fast form. A single H+ is taken up in the switch to the "slow" conformer, which has apparent pK approximately 5.9; low pH also stabilizes part of the pseudoknot structure melting at approximately 62 degrees C. Mg2+ and H+ appear to direct alpha mRNA folding by relatively small (10 to 100-fold) differences in their affinities for alternative conformers. K+ has very little effect on the conformational equilibrium, but at high concentrations accelerates interconversion between the conformers. The alpha mRNA conformational switch is similar in its slow kinetics, large activation energy, and Mg2+ dependence of the equilibrium constant to slow steps in the folding of tRNA, group I introns, and RNase P RNA tertiary structures, though it differs from these in the association of a single Mg2+ with the rate-limiting step.

Base Sequence↗
PubMed1995

A conformational switch in a regulated mRNA involves tertiary structure.

The E. coli alpha operon mRNA is autogenously regulated by binding the repressor ribosomal protein S4. Repression occurs via a novel mechanism in which S4 traps the mRNA in a conformation that prevents formation of the complete initiation complex. The conformations have similar stabilities but separated by a high activation energy which is a criterion for a conformational switch. The conformation switch is likely to involve alteration in tertiary structure as indicated by gel electrophoresis and thermal denaturation experiments. It was found that Mg2+ stabilizes the repressed form and tertiary structure, and H+ stabilizes the translated form and have complicated effects on tertiary structure.

Bacterial Proteins↗
PubMed1994

Thermodynamics of folding a pseudoknotted mRNA fragment.

A sequence in the leader and first gene of the Escherichia coli alpha mRNA folds into a complex pseudoknot structure that is required for binding of a translational repressor. The thermal denaturation of a 112 nt RNA containing this structure has been followed by calorimetry and UV hyperchromicity. To determine the partially folded intermediates in unfolding, the denaturation of 13 mutants and of several fragments with successive deletions of helices were investigated as well. An unfolding pathway with seven states is proposed as the simplest mechanism that accounts for the data, and has several implications. (1) The lowest temperature transition appears only in the presence of moderate concentrations of Mg2+ or high concentrations of K+ (delta H approximately 45 kcal/mol), and is the unfolding of tertiary structures, rather than secondary structure. Under some conditions it is destabilized by increasing salt concentration. (2) Two of the intermediates unfolding at higher temperature must have non-canonical or tertiary interactions in addition to the known secondary structure. (3) Two alternative structures compete for formation of the complete pseudoknot, and form as the pseudoknot unfolds. Thus structures not present in the completely folded pseudoknot affect the overall thermodynamics, and probably the kinetics, of unfolding. (4) Approximately 16 kcal/mol of free energy is required to completely expose the coding region to ribosomes at 37 degrees C, though approximately 6.5 kcal/mol is regained by refolding of upstream regions after the pseudoknot is unfolded. The substantial energy needed to unfold the pseudoknot may affect the rate of translation from this ribosome binding site. A simple model of RNA folding in which an optimum secondary structure forms first, followed by tertiary interactions that further stabilize the secondary structure, does not hold in this RNA.

Base Sequence↗
PubMed1994

Stabilization of RNA structure by Mg ions. Specific and non-specific effects.

The stabilities of three different RNA fragments have been measured as a function of Mg2+ concentrations, and are interpreted in terms of two different models of Mg(2+)-RNA interaction. The models presume either tight binding of ions to specific site(s) in a folded RNA, or non-specific, electrostatic binding to both folded and unfolded forms; qualitatively different predictions are made for the melting temperature dependence on ion concentration. Three different modes of Mg2+ interactions with RNA structures are distinguished. Hairpins are stabilized by completely non-specific binding, with affinities the same as or weaker than those for single strand and duplex homopolymers binding Mg2+. In contrast, a ribosomal RNA tertiary structure is stabilized by specific binding of a single ion; since no other di- or trivalent ion is as effective as Mg2+, direct coordination of Mg2+ to the RNA structure is probably taking place. A third class of sites is best analyzed by the site-specific model, but any di- or trivalent ion is as effective as Mg2+; the ion(s) are probably localized in a region of unusually high charge density. The magnitude of a Mg(2+)-induced shift in RNAtm is itself not diagnostic of specific site binding or RNA tertiary structure, since large shifts can be observed for all three interaction modes.

Binding Sites↗
PubMed1993

Ribosome initiation complex formation with the pseudoknotted alpha operon messenger RNA.

The Escherichia coli alpha mRNA has a complex pseudoknot secondary structure that forms the recognition site for a translational repressor, ribosomal protein S4, and also encompasses the regulated ribosome binding site. To find out whether the pseudoknot is a stable structure under the conditions of ribosome initiation complex formation, thermal denaturation of the RNA was monitored by calorimetry and ultraviolet light hyperchromicity. The secondary structure formed by the coding region melts in a single transition and has a stability of -7.4 kcal/mol at 37 degrees C (5 mM-Mg2+, 100 mM-Na+, pH 7.0). A broad transition with tm approximately 38 degrees C may be a rearrangement of pseudoknot secondary or tertiary structure. Using reverse transcriptase primer extension assays ("toeprints") to measure the kinetics of ternary 30 S subunit-tRNAf(met)-alpha mRNA translational initiation complex formation, we find a fast and a slow phase in the reaction. The fraction reacting rapidly is sensitive to temperature and mutations in the mRNA. We interpret these results in terms of "active" and "inactive" mRNA conformations that are trapped by 30 S subunits and react rapidly or slowly with tRNAf(met), respectively; the active form is predominant above 37 degrees C. The binary 30 S-mRNA complex in the inactive form stops MMLV reverse transcriptase near the 3' edge of the pseudoknot structure, apparently by stabilizing the pseudoknot. We propose the following mechanism for translational initiation with the alpha mRNA. The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNAf(met). The inactive to active transition of the pseudoknot, which may be related to the 38 degrees C transition seen in melting experiments, is required for tRNAf(met) to pair with the anticodon and is rate-limiting for initiation complex formation at lower temperatures. A novel feature of this proposal is that the mRNA structure affects a kinetic step in initiation complex formation, as well as ribosome binding affinity.

Base Sequence↗