Unstructured N terminus of the RNA polymerase II subunit Rpb4 contributes to the interaction of Rpb4.Rpb7 subcomplex with the core RNA polymerase II of Saccharomyces cerevisiae.
Two subunits of eukaryotic RNA polymerase II, Rpb7 and Rpb4, form a subcomplex that has counterparts in RNA polymerases I and III. Although a medium resolution structure has been solved for the 12-subunit RNA polymerase II, the relative contributions of the contact regions between the subcomplex and the core polymerase and the consequences of disrupting them have not been studied in detail. We have identified mutations in the N-terminal ribonucleoprotein-like domain of Saccharomyces cerevisiae Rpb7 that affect its role in certain stress responses, such as growth at high temperature and sporulation. These mutations increase the dependence of Rpb7 on Rpb4 for interaction with the rest of the polymerase. Complementation analysis and RNA polymerase pulldown assays reveal that the Rpb4.Rbp7 subcomplex associates with the rest of the core RNA polymerase II through two crucial interaction points: one at the N-terminal ribonucleoprotein-like domain of Rpb7 and the other at the partially ordered N-terminal region of Rpb4. These findings are in agreement with the crystal structure of the 12-subunit polymerase. We show here that the weak interaction predicted for the N-terminal region of Rpb4 with Rpb2 in the crystal structure actually plays a significant role in interaction of the subcomplex with the core in vivo. Our mutant analysis also suggests that Rpb7 plays an essential role in the cell through its ability to interact with the rest of the polymerase.
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Vinaya
V
Sampath
Bindu
B
Balakrishnan
Jiyoti
J
Verma-Gaur
Silvia
S
Onesti
Parag P
PP
Sadhale
D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers; D009154:Mutation; D012319:RNA Polymerase II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C423309:RPB4 protein, S cerevisiae