Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3' cleavage.
RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring approximately 26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.
Ryan
Kevin
K
Department of Biological Sciences, Columbia University, 117A Fairchild Building, New York, NY 10027, USA.
Murthy
Kanneganti G K
KG
Kaneko
Syuzo
S
Manley
James L
JL
D024509:3' Flanking Region / Q000502:physiology; D000818:Animals; D016384:Consensus Sequence; D005982:Glutathione Transferase / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid / Q000502:physiology; D013329:Structure-Activity Relationship
D021122:Protein Subunits; D012322:RNA Precursors; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D012319:RNA Polymerase II