Mechanism of Poly(A) Polymerase: Structure of the Enzyme-MgATP-RNA Ternary Complex and Kinetic Analysis

Balbo, Paul; Bohm, Andrew

doi:10.1016/j.str.2007.07.010

Cited by 77 publications

(148 citation statements)

References 60 publications

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“…Similarly, C-2 modified ATP analog, 2-amino-ATP, is used efficiently as a substrate by bovine PAP [26]. This is in contrast to a recent crystal structure of yPAP [27], which suggests that substrate specificity of PAP discriminates against groups at C2 of purines as a mechanism for selection of ATP over GTP.…”

Section: Discussioncontrasting

confidence: 59%

Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

2008

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The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) was examined with various ATP analogues of clinical relevance. The triphosphate derivatives of cladribine (2-CldATP), clofarabine (Cl-F-ara-ATP), fludarabine (F-ara-ATP), and related derivatives were incubated with yPAP and 32 P-radiolabeled RNA oligonucleotide primers in the absence of ATP to assay polyadenylation. While 2-Cl-ATP resulted in primer elongation, ara-ATP and F-ara-ATP were poor substrates for yPAP. In contrast, the triphosphate derivatives of cladribine (2-Cl-dATP), clofarabine (Cl-F-ara-ATP) and its corresponding deoxyribose derivative (Cl-F-dATP) were substrates and caused chain termination in the absence of ATP. We further investigated whether analog incorporation at the 3′-terminus of RNA primers negatively impacts polyadenylation with ATP by generating RNA oligonucleotides containing either a terminal clofarabine, Cl-F-dAdo, or cladribine residue. Incorporation of any of these analogs blocks the ability of yPAP to extend RNA past the analog site, impeding the addition of a poly(A)-tail. To determine whether modified ATP analogues exhibit a concentration dependent effect on polyadenylation, poly(A)-tail synthesis by yPAP with modified ATP analogues in combination with a constant level of ATP was also examined. With all the ATP analogues assayed in these studies, there was a significant reduction in poly(A)-tail length with increasing amounts of analog triphosphate. Taken together, our results suggest that polyadenylation inhibition may be a component in the mechanism of action of adenosine analogues.

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Section: Discussioncontrasting

confidence: 59%

Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

2008

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“…The structure of Pap1p in a ternary complex with MgATP and an oligo(A) shows that only the last 3 nucleotides of the pre-mRNA substrate is bound tightly by the enzyme (Fig. 5A) [187].…”

Section: Poly(a) Polymerase (Pap1p)mentioning

confidence: 99%

“…PAP is an induced-fit enzyme, and this induced-fit behavior has an important role in defining the substrate specificity of the enzyme [187][188][189][190]. The N-terminal domain undergoes a large movement upon the formation of the ternary complex with the MgATP and pre-mRNA substrates, whereas MgGTP cannot induce this active site closure.…”

Section: Poly(a) Polymerase (Pap1p)mentioning

confidence: 99%

Protein factors in pre-mRNA 3′-end processing

Mandel

Bai

Tong

2007

Cell. Mol. Life Sci.

364

482

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Most eukaryotic mRNA precursors (pre-mRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3′-end. Processing at the 3′-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, and more than 20 proteins have been identified for the yeast complex. The 3′-end processing machinery also has important roles in transcription and splicing. The mammalian machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factor I (CF I m ), and cleavage factor II (CF II m ). Additional protein factors include poly(A) polymerase (PAP), poly(A) binding protein (PABP), symplekin, and the C-terminal domain (CTD) of RNA polymerase II largest subunit. The yeast machinery includes cleavage factor IA (CF IA), cleavage factor IB (CF IB), and cleavage and polyadenylation factor (CPF).

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“…These specific interactions occur in conjunction with a sequential rearrangement of the binding pocket to accommodate the growing 3′ end and the incoming nucleotide and thus ensure the correct synthesis of CCA synthesis [35,36]. Recently reported ternary structures of PAPs and TUTases with nucleotide and primer similarly suggest that specificity for nucleotide selection results from interactions of the incoming nucleotide with active site amino acids as well as with the RNA primer [31,37].…”

Section: Structure Of Active Sites In Cca-adding Enzymesmentioning

confidence: 99%

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

Martín

Doublié

Keller

2008

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Poly(A) polymerases were identified almost fifty years ago as enzymes that add multiple AMP residues to the 3′ ends of primer RNAs without use of a template from ATP as cosubstrate and with release of pyrophosphate. Based on sequence homology of a signature motif in the catalytic domain, poly(A) polymerases were later found to belong to a superfamily of nucleotidyl transferases acting on a very diverse array of substrates. Enzymes belonging to the superfamily can add from single nucleotides of AMP, CMP or UMP to RNA, antibiotics and proteins but also homopolymers of many hundred residues to the 3′ ends of RNA molecules.The recently reported structures of several nucleotidyl transferases facilitate the study of the catalytic mechanisms of these very diverse enzymes. Numerous structures of CCA-adding enzymes have now revealed all steps in the formation of a CCA tail at the 3′ end of tRNAs. In addition, structures of poly(A) polymerases and uridylyl transferases are now available as binary and ternary complexes with incoming nucleotide and RNA primer. Some of these proteins undergo significant conformational changes after substrate binding. This is proposed to be an indication for an induced fit mechanism that drives substrate selection and leads to catalysis. Insights from recent structures of ternary complexes indicate an important role for the primer molecule in selecting the incoming nucleotide.

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Mechanism of Poly(A) Polymerase: Structure of the Enzyme-MgATP-RNA Ternary Complex and Kinetic Analysis

Cited by 77 publications

References 60 publications

Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

Polyadenylation inhibition by the triphosphates of deoxyadenosine analogues

Protein factors in pre-mRNA 3′-end processing

Determinants of substrate specificity in RNA-dependent nucleotidyl transferases

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