Yeast Poly(A)
Polymerase
Garrett Thesing '24 and Logan Spiess '23
Contents:
I. Introduction
In eukaryotes, pre-mRNA transcripts are cleaved at a consensus
sequence and a tract of 100 to 250 adenine nucleotides are added to
the 3' end of the nascent transcript. This process is known as
polyadenylation and it is an important step in RNA processing. The
addition of the poly(A) tail helps protect the transcript from
degradation and aids in its export from the nucleus to the cytoplasm
where it can be translated into a polypeptide. The poly(A) tail is
also essential for the formation of the closed loop structure that is
necessary for translation initiation in most eukaryotic mRNA
transcripts. In some mRNA transcripts, there can be multiple
polyadenylation sites. Because many binding sites for regulatory
proteins and RNAs exist in the 3' UTR, alternative polyadenylation in
this region can have a profound effect on gene expression. In some
cases, alternative polyadenylation sites can occur in the coding
region of a gene which can result in the production of distinct
protein isoforms from the same gene.
Of the several proteins that are involved in polyadenylation,
poly(A) polymerase (PAP) is the enzyme which is responsible for
adding a poly(A) tail to the 3' end of pre-mRNA transcripts. It is a
template-independent polymerase which belongs to the DNA polymerase
beta family (Balbo and Bohm). It uses the 3' end of the nascent mRNA
strand as a primer and adds adenine nucleotides non-templated. It
can synthesize the poly(A) tail in a processive manner when other
components of the polyadenylation machinery are bound to the RNA
substrate (Wahle).
II. General Structure
Yeast poly(A) polymerase is 530 amino acids long and is
composed of three domains; the N-terminal
domain (residues 40-190), middle
domain (residues 1-39 and 190-353) and C-terminal
domain (residues 353-530) encircle a substrate binding
cleft where RNA
and ATP bind (Balbo and Bohm)
. The N-terminal domain is homologous to the catalytic palm domain
of other beta polymerases. In the active site, three aspartate
residues (100, 102,
154) coordinate two catalytically-required magnesium
ions (Zhelkovsky et al.). In this structure, Asp154 is
mutated to Ala154 so the second
magnesium ion is missing
. The C-terminal domain plays an important role in holding the RNA
substrate. The middle domain is thought to function similarly to the
fingers domain of template-directed polymerases in that it helps to
properly orient the incoming ATP in the active site (Balbo and
Bohm).
III. ATP Specificity
In order for Poly(A) Polymerase to add adenines to the 3'
end of the pre-mRNA transcript, it must have specificity for ATP
in its active site. Asp100
and Asp 102 interact with a magnesium
ion which in turn interacts with the phopsphates of ATP,
helping to position it in the active site
. There is a non-polar base stacking interaction between the adenine
base base of ATP and the
3'-terminus of the RNA
substrate
. On the opposite side of the base stacking interaction,
Val 234 makes Van der Waals interactions with the adenine
base and ribose of ATP
(Balbo and Bohm). These non-polar interactions promote purine
specificity in the active site. Three water
molecules buried within the complex near the active site
hydrogen bond with N3, N6, and N7 of the adenine
base
. These specific hydrogen bonds promote specificity for ATP over
GTP. The side chains of Thr 304, Met310,
and Ala312 come in close contact with
the C2 of adenine
. This close steric contact further promotes specificity for ATP
over GTP; the amino group at this position in guanine would not fit
between these amino acid residues (Hodis et al.)
IV. RNA Binding
4 nucleotides at the 3' end of the Poly(A) RNA make
contact with PAP. The -4 nucleotide
(5'-most residue) makes contact with the surface of the enzyme
while the -3, -2, and -1
(3'-terminal residue) nucleotides are in contact with the interior
of the enzyme
. The -1 nucleotide is
positioned between Val141 and the
incoming ATP
. Several hydrogen bonds form between the -1 nucleotide
and amino acid residues of the enzyme. The 2' hydroxyl forms a hydrogen
bond with the side
chain hydoxyl of Tyr87
and the phosphate group oxygen forms a hydrogen bond with
Asn226
. Additionally, the -1 nucleotide is bound in the enzyme
through an interaction between its 3' hydroxyl and a catalytic
magnesium ion which is coordinated by Asp154 (Balbo and Bohm). As
mentioned above, Asp154 is mutated to Ala154 so the magnesium ion
is not present in this structure. Overall, the positioning of the
Poly(A) RNA within the enzyme ensures that the incoming adenine
nucleotide can be effectively added to the 3' end.
V. Closed Ternary Complex
The absence of the second catalytically-required magnesium
ion inhibits the catalytic activity of the enzyme but does not
affect binding of the RNA and ATP substrates. When both the RNA
and ATP substrates are bound in the active site, poly(A)
polymerase adopts its 'closed' substrate-bound ternary complex.
There are two defined hinge regions in PAP: one between the
N-terminal and middle domains (residues around 40 and 190) and one
between the middle and C-terminal domains (residues around
353)(Balbo and Bohm). When PAP adopts its 'closed' conformation,
movement of the domains about these hinge regions is observed:
both the N-terminal and C-terminal domains rotate inward toward
the middle domain.
VI. References
Balbo, Paul B, and Andrew Bohm. Mechanism of poly(A) polymerase: structure of the enzyme-MgATP-RNA ternary complex and kinetic analysis. Structure (London, England : 1993) vol. 15,9 (2007): 1117-31. doi:10.1016/j.str.2007.07.010.
Hodis, Eran, Michal Harel, David Canner, Joel L. Sussman, OCA, Alexander Berchansky, Jaime Prilusky, David S. Goodsell, Eric Martz. Poly(A) Polymerase. Proteopedia. https://proteopedia.org/wiki/index.php/Poly%28A%29_Polymerase.
Zhelkovsky, Alexander, Steffen Helming, Andrew Bohm, Claire Moore. et al. Mutations in the Middle Domain of Yeast Poly(a) Polymerase Affect Interactions with RNA but Not ATP. RNA. vol. 10, no. 4, 2004, pp. 558-564., https://doi.org/10.1261/rna.5238704
Elmar Wahle. Poly(A) Tail Length Control Is Caused by Termination of Processive Synthesis. Journal of Biological Chemistry. vol. 270, Issue 6, 1995, pp. 2800-2808, https://doi.org/10.1074/jbc.270.6.2800.
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