Effect of Transcriptional Parameters on the Folding of the Tetrahymena Group I Intron
Koduvayur, Sujatha P
Woodson, Sarah A
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Differential elongation rates and pausing patters of polymerases are known to affect co-transcriptional folding of RNAs. The mechanism of coupling is not well studied. This study evaluates whether these factors contribute to the 20-50 fold splicing enhancement seen in vivo for the Tetrahymena group I intron. The splicing rates of T7 and Escherichia coli (E. coli) RNA polymerase (RNAP) transcripts were compared in vitro and in E. coli. T7 RNAP is a rapid and highly processive polymerase, while E. coli RNAP elongates transcripts more slowly. In a collaborative study with Scott Jackson, the splicing of transcripts of eukaryotic polymerase I and II (pol I and pol II) were compared in Saccharomyces cerevisiae. As the stability of nascent structures can be altered by changing the sequence of transcribed RNA, I have also studied the effect of rRNA exons on the folding of this RNA by comparing the splicing rates of pre-RNAs with E. coli and Tetrahymena thermophila domain IV rRNA exons in vitro and E. coli. In yeast, S. cerevisiae and T. thermophila rRNA, and GFP mRNA exon sequences were compared. My E. coli and in vitro comparative study reveals that co-transcriptional folding is the reason for the rate enhancement seen in vivo for this RNA. For E. coli RNAP transcripts this is effected by the template-dependant, site-specific pausing of the polymerase along the template. For the pre-RNA with Tetrahymena rRNA exons this is effected by the low stability of nascent structures that enable rapid rearrangement of non-native structures into native ones. In yeast, we find that both the polymerase and the RNA processing events affect folding of this intron. Pol I transcripts splice 10-fold better than their pol II counterparts and mRNA processing events retard splicing of short pol II transcripts by 10-fold. Moreover, in yeast, only mutations that increase the fraction of misfolded intermediates are rescued but not those that destabilize the native structure. This folding facilitation is however not dependant on the presence of longer rRNA exons in yeast as it is in E. coli. This might be indicative of a different folding facilitatory mechanism in yeast from that seen in E. coli.