Biochemische Charakterisierung der siRNA-vermittelten Erkennung ...

Summary
RNA interference (RNAi) is a highly specic mechanism to control gene expression on the
posttranscriptional level. It is found in all higher organisms including humans and comprises
the sequence-dependent recognition and binding of a target mRNA by the ribonucleoprotein
complex RNA-induced silencing complex (RISC). Consequently, mRNA translation is blocked
either by endonucleolytic cleavage or by steric hindering of the translation machinery. The
Argonaute2 protein (hAgo2) is the key component of human RISC and associates with a
ribonucleic acid portion, the guide RNA. The enzyme is composed of four domains, namely
the N-terminal, PAZ, Mid and PIWI domains. Its ability to bind any given guide RNA makes
it a universal molecular tool of cells. The aim of this PhD thesis was the biochemical and
kinetic characterisation of recombinant hAgo2 in vitro.
The initial goal was to obtain sucient amounts of enzymatically active recombinant hAgo2.
Therefore, ve dierent strategies for the expression and purication were established and
tested in parallel. Poor solubility and incomplete translation of the fusion proteins were the
major problems encountered. Nevertheless, the isolation of hAgo2 bearing an N-terminal GSTtag
under non-denaturing conditions could be achieved. The protein was puried in milligram
amounts and showed high enzymatic activity. All tested constructs were able to cleave target
RNA in a characteristic, sequence-specic manner, whereas a C-terminally modied construct
exhibited a drastic decrease in turnover as compared to proteins with non-modied C-termini.
This suggests that the tag disrupts the correct protein folding and emphasizes similarities
between the domain structures within prokaryotic and eukaryotic Ago proteins. Furthermore,
insight concerning the optimal Mg 2+ concentration for the cleavage of target RNA as well
as the oligomerisation behaviour of hAgo2 could be gained. hAgo2 was found to possess the
tendency to aggregate, which can be induced by high temperature. This process can almost
completely be abrogated by the addition of RNA.
The main focus of this thesis was the biochemical and kinetic characterisation of the siRNAmediated
target RNA cleavage by recombinant hAgo2. The process could be broken down into
four steps (loading of hAgo2 with an siRNA, target RNA recognition and binding, its cleavage
and release of the cleaved products). Consequently, these four steps were investigated individually
by performing appropriate experiments. In a rst set of experiments, hAgo2 loading
was analysed. For this matter, a uorescence-based system was developed and used to observe
the binding reaction of hAgo2 with single- and double-stranded siRNA under steady and
pre-steady state conditions. It could be shown for the rst time, that recombinant hAgo2 is
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