A pre-mRNA contains two identical 5-splice sites, one being located 300 nucleoti

Question

A pre-mRNA contains two identical 5-splice sites, one being located 300 nucleotides downstream from the first one. In an in vitro experiment, a radioactive RNA with these two 5-splice sites is incubated with a nuclear extract competent for splicing, ATP, and MgCl In vitro 50%! 50% 3" AG 5 99% n vivo Both sites are spliced; 50% at the upstream splice site and 50% at the downstream splice site. However, in vivo the upstream splice site is used 99% of the time. Considering the abova iadiam,explain why this occurs

Explanation / Answer

In in vitro splicing is not dependent of its original positioning since there are no obligatory order in the excision and rate in in vitro is much slower that which occurs in the cell or in vivo. it occurs without nuclear compartmentalization,, allowing the machinery to be decoupled form the trabscriptional machinery which probably leads to decrease in splicing rate as spliceosome recruitement occurs cotranscriptionally. The kinetic analysis of in vitro splicing take adantage of pre-mRNA substrate required and excess of the splicing factors contained within the nuclear extract.the appearance of the product can be determined with a first order reaction.

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