Processivity of the Saccharomyces cerevisiae poly(A) polymerase requires interactions at the carboxyl-terminal RNA binding domain.
The interaction of the Fip1 subunit of polyadenylation factor I with the Saccharomyces cerevisiae poly(A) polymerase (PAP) was assayed in vivo by two-hybrid analysis and was found to involve two separate regions on PAP, located at opposite ends of the protein sequence. In vitro, Fip1 blocks access of the RNA primer to an RNA binding site (RBS) that overlaps the Fip1 carboxy-terminal interaction region and, in doing so, shifts PAP to a distributive mode of action. Partial truncation of this RBS has the same effect, indicating that this site is required for processivity. A comparison of the utilization of ribo- and deoxyribonucleotides as substrates indicates the existence on PAP of a second RBS which recognizes the last three nucleotides at the 3' end of the primer. This site discriminates against deoxyribonucleotides at the 3' end, and interactions at this site are not affected by Fip1. Further analysis revealed that the specificity of PAP for adenosine is not simply a function of the ATP binding site but also reflects interactions with bases at the 3' end of the primer and at another contact site 14 nucleotides upstream of the 3' end. These results suggest that the unique specificity of PAP for ribose and base, and thus the extent and type of activity with different substrates, depends on interactions at multiple nucleotide binding sites.
Zhelkovsky
A
A
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111-1800, USA.
Helmling
S
S
Moore
C
C
D001665:Binding Sites; D017931:DNA Primers; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D039221:mRNA Cleavage and Polyadenylation Factors
D017931:DNA Primers; D012331:RNA, Fungal; D016601:RNA-Binding Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D011062:Polynucleotide Adenylyltransferase