Chain termination and inhibition of Saccharomyces cerevisiae poly(A) polymerase by C-8-modified ATP analogs.
The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. 32P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.
Chen
Lisa S
LS
Department of Chemistry and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60208-3113, USA.
Sheppard
Terry L
TL
D000241:Adenosine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011061:Poly A / Q000737:chemistry; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry*; D011487:Protein Conformation; D012313:RNA / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors
D017931:DNA Primers; D009841:Oligonucleotides; D011061:Poly A; D012313:RNA; D000255:Adenosine Triphosphate; D011062:Polynucleotide Adenylyltransferase; D000241:Adenosine