AbstractThe kink-turn in RNA is a secondary structural motif which produces a tight kink in the axis of the RNA helix. It exists in a dynamic equilibrium between the folded and the unfolded state in free solution. The population of the folded kink-turn species can be increased by the addition of metal ions which bind to and stabilise the kink-turn structure. Standard kink-turn RNAs differ in their ability to undergo metal-ion induced folding. For example, the ribosomal kink-turn, HmKt-7, readily folds into the kinked conformation in metal ions. The AfboxC/D snoRNA kink-turn, on the other hand, is unable to fold in metal ions and requires the binding of the L7Ae protein to fold. The reason for this variation lies in the kink-turn RNA sequence. Certain positions, namely G1b:A1n and A2b:G2n, are critical for metal ion-induced kink-turn folding and form the basis for the classification of a RNA sequence as a standard kink-turn motif. The 3b:3n position has also been identified as being very important for metal-ion induced folding of kink-turns with different base pairs providing varying degrees of folding ability. U3b:U3n allows moderate folding of the modified HmKt-7 kink-turn in the presence of metal ions. The AfboxC/D kink-turn, which has U3b:U3n, is thus expected to fold. However, this is not the case leading to this study of other positions in the kink-turn RNA sequence which could potentially affect its folding.
The -1b:-1n, 3b:3n and 4b:4n positions of the Haloarcula marismortui Kt-7 and Archeoglobus fulgidus box C/D kink-turn RNAs have been studied in this work. The effect of modifying these positions on the ability of the kink-turns RNA to fold on the addition of metal ions has been investigated. RNA folding was studied using the techniques of fluorescence resonance energy transfer (FRET) and gel electrophoresis. The systematic exchange of sequence elements between the HmKt-7 and AfboxC/D kink-turn RNAs has revealed the importance of sequence on kink-turn RNA folding. The -1b:-1n, 3b:3n and 4b:4n positions all have an additive effect on the folding ability of kink-turns with the most profound effect exerted by the -1b:-1n position. In general, HmKt-7 has selected sequence elements that are C-1b:G-1n, A3b:G3n and C4b:G4n, which promote the metal ion-induced folding of the kink-turn. On the other hand, AfboxC/D, despite having the moderate folding U3b:U3n sequence element, has G4b:C4n and, in particular, G-1b:C-1n which inhibits the folding of the RNA into the kink-turn structure in metal ions alone.
Furthermore, the N6-methylation of adenine is a naturally occurring modification in cellular RNA which is also prevalent in the kink-turn regions of RNA. Hence, the effect of this modification on kink-turn RNA folding in the presence of metal ions, as well as the L7Ae protein, was studied by FRET. Substitutions in the various positions of the HmKt-7 kink-turn RNA sequence have revealed a differential effect on kink-turn folding. While N6-methyladenine in the 1n position completely prevents metal ion and protein-induced folding of the kink-turn, the 2b and 3b positions are more tolerant although folding is impaired to some extent. These results provide some valuable insights to the effect that this naturally occurring modification can have on kink-turn RNA folding should any be found.
On the whole, the folding characteristics of kink-turns as determined by their sequence, is in adoption to their function. For example, ribosomal and riboswitch kink-turns have generally selected sequence elements that allow folding into the kinked conformation in metal ions alone which may be in accordance to their biological duty requiring free formation of the kink-turn structure. On the other hand, box C/D snoRNA kink-turns have selected sequence elements that render them unable to fold in metal ions alone but requiring the binding of the L7Ae-type protein to fold which is actually the first step in the assembly of the biologically active box C/D snoRNP complex. Nevertheless, naturally occurring sequence modifications, such as N6-adenine methylation, can also alter the folding capability of kink-turns. A recently found example suggests that the naturally occurring N6-methyladenine modification in the 1n position of the box C/D kink-turn RNA sequence can prevent the binding of the 15.5k protein, and hence prevent the assembly of the box C/D snoRNP complex. Thus, overall, in addition to sequence determinants, modifications such as N6-adenosine methylation can modulate RNA structure and consequently regulate RNA function.
|Date of Award||2017|
|Supervisor||David Lilley (Supervisor)|
- fluorescence resonance energy transfer
Sequence Dependence of the Folding of Kink-Turns in RNA
Ashraf, S. (Author). 2017
Student thesis: Master's Thesis › Master of Science