Julia Lewis and Mithila Rajagopal
Mentor: Professor White
L30e is an autoregulatory ribosomal protein. In Saccharomyces cerevisiae, L30e binds to its RNA transcript to inhibit splicing and to its mRNA to repress translation. L30e’s secondary structure consists of six alpha helices and 4 beta sheets. The RNA secondary structure is similar to the 3-D folding of a protein, consisting of motifs such as hairpins, pseudoknots, internal loops, etc. L30e RNA contains a kink-turn motif that is present in both the L30e messenger and ribosomal RNA. The kink-turn causes a sharp bend in the RNA double helix. The kink-turn motif is a canonical stem of Watson-Crick base pairs, three unpaired nucleotides, and a non-canonical stem having two sheared G:A pairs.
The goal of our lab’s research is to understand how some irregular structural features relate to L30e recognition and binding. Previous work in the lab consisted of synthesizing RNA and protein variants to observe the effect of specific mutations on in vitro and in vivo RNA-protein interaction. The RNA mutants were subjected to electrophoresis on a polyacrylamide gel. The rate at which the RNA moves through the gel is related to its shape. The most bent or compact RNA molecule will pass through a gel the fastest because it offers less resistance to the gel. By comparing the relative gel mobilities of RNA mutants, the extent to which each mutation affects the formation of a kink turn can be determined. This makes it possible to determine which mutants assume specific secondary and tertiary structures.
To study the RNA we first started with bacteria streaks of E. coli which were grown from already synthesized plasmids BPAU, BP3, KTAU and KT22. The BP (base paired) plasmid vectors contain DNA in which nucleotides are perfectly base paired. The KT (kink-turn) contain DNA which get transcribed into RNA in which nucleotides are not base paired resulting in the formation of a kink-turn. Mutations were produced in the KT bacteria to study its effects on the kink-turn of the RNA. The attempted mutation, KTAU A12U (12th nucleotide changed from an adenine to a uridine) showed very poor growth. The remaining bacterial streaks of BP and KT were used in the isolation of DNA both by small scale (3ml) and large scale (6ml) techniques. The small-scale resulted in lower yields but equal purity as indicated by analytical gels and UV-vis spectroscopy. RNA was synthesized from this DNA by first performing a restriction digest using restriction enzyme SpeI which cleaved the circular DNA making it linear and suitable for transcription into RNA. After transcription, agarose gels were run to estimate the quantity and purity of RNA obtained. This research hopes to perform study the effects of various mutations on the tertiary structure of the kink-turn motif of the RNA using electophoresis gel experiments (polyacrylamide gels), sedimentation experiments and atomic Force Microscopy (AFM) which would show how bent or compact the RNA is (to what extent the kink-turn is affected).