Hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16S rRNA in the 30S ribosome

<p>Abstract</p> <p>Background</p> <p>Conformational flexibility in structured RNA frequently is critical to function. The 30S ribosomal subunit exists in different conformations in different functional states due to changes in the central part of the 16S rRNA. We are interested in evaluating the factors that might be responsible for restricting flexibility to specific parts of the 16S rRNA using biochemical data obtained from the 30S subunit in solution. This problem was approached taking advantage of the observation that there must be a high degree of conformational flexibility at sites where UV photocrosslinking occurs and a lack of flexibility inhibits photoreactivity at many other sites that are otherwise suitable for reaction.</p> <p>Results</p> <p>We used 30S x-ray structures to quantify the properties of the nucleotide pairs at UV- and UVA-s⁴U-induced photocrosslinking sites in 16S rRNA and compared these to the properties of many hundreds of additional sites that have suitable geometry but do not undergo photocrosslinking. Five factors that might affect RNA flexibility were investigated – RNA interactions with ribosomal proteins, interactions with Mg²⁺ions, the presence of long-range A minor motif interactions, hydrogen bonding and the count of neighboring heavy atoms around the center of each nucleobase to estimate the neighbor packing density. The two factors that are very different in the unreactive inflexible pairs compared to the reactive ones are the average number of hydrogen bonds and the average value for the number of neighboring atoms. In both cases, these factors are greater for the unreactive nucleotide pairs at a statistically very significant level.</p> <p>Conclusion</p> <p>The greater extent of hydrogen bonding and neighbor atom density in the unreactive nucleotide pairs is consistent with reduced flexibility at a majority of the unreactive sites. The reactive photocrosslinking sites are clustered in the 30S subunit and this indicates nonuniform patterns of hydrogen bonding and packing density in the 16S rRNA tertiary structure. Because this analysis addresses inter-nucleotide distances and geometry between nucleotides distant in the primary sequence, the results indicate regional and global flexibility of the rRNA.</p

A 16S rRNA–tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome

Ribosome complexes containing deacyl-tRNA(1)(Val) or biotinylvalyl-tRNA(1)(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo(5)U nucleoside at position 34 in the tRNA(1)(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo(5)U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450–5454]. However, in complexes containing deacyl-tRNA(1)(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m(5)C967 of 16S rRNA and cmo(5)U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA(1)(Val) –70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state

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

Bacterial tRNAs frequently have 4-thiouridine (s4U) modification at position 8, which is adjacent to the C13-G22-m7G46 base triple in the elbow region of the tRNA tertiary structure. Irradiation with light in the UVA range induces an efficient photocrosslink between s4U8 and C13. The temperature dependence of the rate constants for photocrosslinking between the s4U8 and C13 has been used to investigate the tRNA conformational energy and structure in Escherichia coli tRNAVal, tRNAPhe, and tRNAfMet under different conditions. Corrections have been made in the measured rate constants to compensate for differences in the excited state lifetimes due to tRNA identity, buffer conditions, and temperature. The resulting rate constants are related to the rate at which the s4U8 and C13 come into the alignment needed for photoreaction; this depends on an activation energy, attributable to the conformational potential energy that occurs during the photoreaction, and on the extent of the structural change. Different photocrosslinking rate constants and temperature dependencies occur in the three tRNAs, and these differences are due both to modest differences in the activation energies and in the apparent s4U8–C13 geometries. Analysis of tRNAVal in buffers without Mg2+ indicate a smaller activation energy (∼13 kJ mol−1) and a larger apparent s4U8–C13 distance (∼12 Å) compared to values for the same parameters in buffers with Mg2+ (∼26 kJ mol−1 and 0.36 Å, respectively). These measurements are a quantitative indication of the strong constraint that Mg2+ imposes on the tRNA flexibility and structure

Efficiency and pattern of UV pulse laser-induced RNA–RNA cross-linking in the ribosome

Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10–40 mJ for a 1 cm(2) area, corresponding to fluences of 4.5 to 18 × 10(9) W m(–2), to determine strand breakage yields and the frequency and pattern of RNA–RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 × 10(–3) over the incident intensity range studied. The yield for RNA–RNA cross-linking was 3 × 10(–4) cross-links/photon at the intensity of 4.5 × 10(9) W m(–2), an ∼4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation