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

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.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.There is a long-standing interest in the connection between the interior packing arrangement and conformational dynamics of proteins. Richards [1] recognized early the importance of packing in the protein interior and summarized methods for calculating packing density; he further speculated that irregular packing, if it results in gaps or cavities, could lead to specific conformational motions. This idea lacked support for some time, given evidence that efficient packing was important to protein stability