Internucleotide Movements during Formation of 16 S rRNA–rRNA Photocrosslinks and their Connection to the 30 S Subunit Conformational Dynamics

Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s4U8–C13 photocrosslinking

“…These measurements indicated a correlation between the reciprocal C19-C19 internucleotide distances and the photocrosslinking frequencies; the correlation coefficients have values between 0.39 to 0.7 using the Thermus thermophilus 30S structure (Wimberly et al 2000) or two E. coli 30S structures (Schuwirth et al 2005). These correlations and the comparison of the data for the UV-and UVA-s 4 U-induced crosslinks are consistent with reaction mechanisms that involve transient movements, which occur during the excited electronic state lifetime of the nucleobase, to bring the pairs of nucleobases from their equilibrium positions to the arrangement needed for photoreaction, rather than mechanisms in which there are stable alternate local conformations at the crosslinking sites (Huggins et al 2005). An important feature of this mechanism is that the transition state for the photoreaction is reached without the system having to go through any higher potential energy.…”

“…Differences in the observed rate constants are not accounted for by the activation energies alone. From a study of the connections between internucleotide geometry and photocrosslinking frequency in ribosomal RNA, we expected that photocrosslinking rates would depend on the internucleotide distance (Huggins et al 2005). This dependence is probably due to the fact that the excited state lifetime of the s 4 U limits the extent of the conformational excursions.…”

“…It will be seen that the preexponential term varies by >20-fold under different conditions, and it is very unlikely that this difference is due to differences in the entropy change (see Discussion). On the other hand, we had already observed that photocrosslinking frequencies tend to be larger for sites in the rRNA where the internucleotide distance is smaller (Huggins et al 2005). Therefore the expression for the crosslinking rate constant should contain the inverse of the s 4 U8-C13 internucleotide distance to incorporate this dependence.…”

“…It would be reasonable that the rate constant will actually depend on a combination of changes in the distance and angle between the photoreactive bonds. Dependence on a combination of distance and angle was not used here because, in the statistical analysis of the factors for the crosslinking frequency in the rRNA, the internucleotide angle did not show a correlation to the frequency (Huggins et al 2005).…”

“…For the 16S rRNA it is likely that local flexibility, determined by hydrogen bonding and packing density, is responsible for inhibition at many potential sites and the modulation of the frequency at the sites where photocrosslinks occur (Huggins et al 2005; W. Huggins, K. Nanda, S.K. Ghosh, T. Shapkina, and P. Wollenzien, unpubl.).…”