CaMKIIalpha 3' untranslated region-directed mRNA translocation in living neurons: visualization by GFP linkage.

The CaMKIIalpha mRNA extends into distal hippocampal dendrites, and the 3' untranslated region (3'UTR) is sufficient to mediate this localization. We labeled the 3'UTR of the CaMKIIalpha mRNA in hippocampal cultures by using a green fluorescent protein (GFP)/MS2 bacteriophage tagging system. The CaMKIIalpha 3'UTR formed discrete granules throughout the dendrites of transfected cells. The identity of the fluorescent granules was verified by in situ hybridization. Over 30 min time periods these granules redistributed without a net increase in granule number; with depolarization there is a tendency toward increased numbers of granules in the dendrites. These observations suggest that finer time resolution of granule motility might reveal changes in the motility characteristics of granules after depolarization. So that motile granules could be tracked, shorter periods of observation were required. The movements of motile granules can be categorized as oscillatory, unidirectional anterograde, or unidirectional retrograde. Colocalization of CaMKIIalpha 3'UTR granules and synapses suggested that oscillatory movements allowed the granules to sample several local synapses. Neuronal depolarization increased the number of granules in the anterograde motile pool. Based on the time frame over which the granule number increased, the translocation of granules may serve to prepare the dendrite for mounting an adequate local translation response to future stimuli. Although the resident pool of granules can respond to signals that induce local translation, the number of granules in a dendrite might reflect its activation history.

An optimal Mg(2+) concentration for kinetic folding of the tetrahymena ribozyme.

Divalent metal ions, such as Mg(2+), are generally required for tertiary structure formation in RNA. Although the role of Mg(2+) binding in RNA-folding equilibria has been studied extensively, little is known about the role of Mg(2+) in RNA-folding kinetics. In this paper, we explore the effect of Mg(2+) on the rate-limiting step in the kinetic folding pathway of the Tetrahymena ribozyme. Analysis of these data reveals the presence of a Mg(2+)-stabilized kinetic trap that slows folding at higher Mg(2+) concentrations. Thus, the Tetrahymena ribozyme folds with an optimal rate at 2 mM Mg(2+), just above the concentration required for stable structure formation. These results suggest that thermodynamic and kinetic folding of RNA are cooptimized at a Mg(2+) concentration that is sufficient to stabilize the folded form but low enough to avoid kinetic traps and misfolding.

Fast folding mutants of the Tetrahymena group I ribozyme reveal a rugged folding energy landscape.

A model for the kinetic folding pathway of the Tetrahymena ribozyme has been proposed where the two main structural domains, P4-P6 and P3-P7, form in a hierarchical manner with P4-P6 forming first and P3-P7 folding on the minute timescale. Recent studies in our laboratory identified a set of mutations that accelerate P3-P7 formation, and all of these mutations appear to destabilize a native-like kinetic trap. To better understand the microscopic details of this slow step in the Tetrahymena ribozyme folding pathway, we have used a previously developed kinetic oligonucleotide hybridization assay to characterize the folding of several fast folding mutants. A comparison of the temperature dependence of P3-P7 folding between the mutant and wild-type ribozymes demonstrates that a majority of the mutations act by decreasing the activation enthalpy required to reach the transition state and supports the existence of the native-like kinetic trap. In several mutant ribozymes, P3-P7 folds with biphasic kinetics, indicating that only a subpopulation of molecules can evade the kinetic barrier. The rate of folding of the wild-type increases in the presence of urea, while for the mutants urea merely shifts the distribution between the two folding populations. Small structural changes or changes in solvent can accelerate folding, but these changes lead to complex folding behavior, and do not give rise to rapid two-state folding transitions. These results support the recent view of folding as an ensemble of molecules traversing a rugged energy landscape to reach the lowest energy state.

Kinetic intermediates trapped by native interactions in RNA folding.

In the magnesium ion-dependent folding of the Tetrahymena ribozyme, a kinetic intermediate accumulates in which the P4-P6 domain is formed, but the P3-P7 domain is not. The kinetic barriers to P3-P7 formation were investigated with the use of in vitro selection to identify mutant RNA molecules in which the folding rate of the P3-P7 domain was increased. The critical mutations disrupt native tertiary interactions within the P4-P6 domain and increase the rate of P3-P7 formation by destabilizing a kinetically trapped intermediate. Hence, kinetic traps stabilized by native interactions, and not simply by mispaired nonnative structures, can present a substantial barrier to RNA folding.