RNA BLOG

The ribosome is a large machinery (~ 20 nm in diameter, 70S sedimentation rate for bacterial ribosomes) and is made of two subunits: a large subunit (~50S) and a small subunit (~ 30S). The large subunit is in turn made of two ribosomal RNA (5S and 23S) and several (~34 proteins) whereas the small subunit has one ribosomal RNA (16S) and ~ 21 proteins. The 23S rRNA is ~ 3000 nucleotides long, and the 16S rRNA is ~ 1500 nucleotides long.

The structures of ribosomal RNA can get very complicated because of the large number of ways in which hairpins and loops can be formed. Predicting these structures requires a combination of both computational methods (in which the most probable secondary structures are determined from estimates of free energy for a given structure) and a variety of experimental techniques.

Oligonucleotide mapping techniques

This technique is useful in identifying exposed single-stranded regions of a folded RNA molecule by hybridization with short synthesized nucleotide chains (also called oligonucleotides) that are complementary to, for instance, the loop regions in RNA.

Folded RNA molecules are confined to one region in space separated by another region by a semi-permeable membrane. On the other side of the partition are radioactive oligonucleotides (~ 5-10 nucleotides long) that can pass through the membrane and bind to RNA molecules, but the RNA molecules, which are much bigger in size, cannot.

At equilibrium, free oligomers are in the same concentration on both sides of the partition. However, the radioactivity on the side with the RNA molecules is larger than the other size because some oligomers will associate with (bind to) RNA if the sequences of oligomers and loop regions are complementary. A measure of the ratio (r_d) of radioactivity from either side gives a measure of the binding or association constant where [X] is the concentration of the RNA-oligomer complex, [O] is the free oligomer concentration on either side, and [RNA] is the concentration of molecules that are not bound to an oligomer.

The ratio

If [RNA] >> [O], then the RNA concentration can be assumed the same before and after mixing and the ratio becomes .

Therefore, a measurement of r_d yields a direct measure of K_a.

All oilgonucleotides will lead to some association since there is always a match at a single base-pair level. Therefore for any oligonucleotide. For oligonucleotides ~ 4 bases long that match an exposed loop region on the RNA the free energy change upon association is substantially larger (by ~ 10-15 k_BT ) than the free energy change from single base-pair matches. This lead to an increase in the association constant by a factor of 10⁴ to 10⁶.

This technique can easily distinguish between two possible conformations of an RNA molecule which have different sequences in their loop regions.

We can also estimate which structure is more probable (i.e. which one has the lower free energy.

The free energy of a hairpin can be broken into two parts, the free energy of forming a loop closed by a single base-pair and the free energy for the base-paired `stem' of the hairpin.

In RNA molecules the most probable loop size consists of ~ 6-7 bases in the loop. Smaller loops are energetically unfavorable as a result of steric hindrances among the bases and atoms of the backbone. Larger loops are entropically unfavorable. The loss of entropy when loops are formed increases with increasing loop size.

for the optimal sized loop closed by a G-C base-pair is ~ 7-8 k_BT in 1M NaCl. In our example we have a loop with 10 bases in structure 1 () and 2 loops with 4 bases each in structure 2 (for each loop).

Note that is a positive quantity; it is unfavorable to make loops relative to the random coil conformation.

The hairpin structures are stabilized when the free energy gain from base-pair formation exceeds the free energy cost of loop formation.

The gain from adding a base-pair to an already existing G-C pair is ~ for adding a G-C base-pair and ~ for adding a A-U base-pair.

Therefore the net change in free energy for structure 1 is

and for structure 2 is