Studying the importance of 2´OH groups of tRNA in the RNase P mediated cleavage
of tRNA precursors
Ramesh Raju Vetukuri
Genetic information in living organisms is stored in the form of deoxyribonucleic acid
(DNA). The information from DNA is copied on to messenger ribonucleic acid (mRNA)
by an enzyme, the RNA polymerase, in a process called transcription. In order to form a
new protein the message has to be translated to amino acids (“building blocks” of the
protein). The mechanism of translation is aided by tRNA (transfer RNA).
Transfer RNAs or tRNAs are formed as longer, non-mature tRNA fragments (called
precursor tRNAs), which have to be cut to shorter mature tRNA before they can aid in
the translation mechanism. RNase P is an enzyme involved in this cutting (also called
cleavage) of tRNA. In this project I studied some of the important nucleotides (“building
blocks” of tRNA) present in tRNA that help in recognition and correct cutting of tRNA
by RNase P. For this study certain changes in the “building blocks” (nucleotides) were
introduced in the precursor tRNA. The changes were made in places that were proposed
to be important for recognition and cutting. This was done by making the tRNA more
DNA-like at different places. I also used a tRNA called mini 3bp that had only 24
nucleotides. It is very short compared to the 70 nucleotides in normal precursor tRNA.
The aim was to see what was the smallest precursor tRNA that could be recognised and
cut by RNase P. I compared the cleavage reaction rate (how fast the cleavage reaction
took place), overall cleavage efficiency and the ability of the RNase P to bind to these
different precursor tRNA to the same reactions with normal precursor tRNA.
The results showed that changes made in these precursor tRNAs did not affect
recognition or cleavage significantly. There was a significant difference concerning the
small precursor tRNA mini 3bp in comparison with normal precursor tRNA. This
difference may be due to the fact that mini 3bp was shorter than to the normal tRNA, and
thus did not look the same to the enzyme. From this I may conclude that mini 3bp has
some important part missing from the whole structure required for recognition and
cleavage.
Degree Project in biology, Uppsala University, vår 2005
Examensarbete i Biologi, 20 p,
Department of Cell and Molecular Biology,
Supervisor: prof. Leif Kirsebom