Crystal structures of the vaccinia virus polyadenylate polymerase heterodimer: insights into ATP selectivity and processivity

Polyadenylation of mRNAs in poxviruses, crucial for virion maturation, is carried out by a poly(A) polymerase heterodimer composed of a catalytic component, VP55, and a processivity factor, VP39. The ATP-gamma-S bound and unbound crystal structures of the vaccinia polymerase reveal an unusual architecture for VP55 that comprises of N-terminal, central or catalytic, and C-terminal domains with different topologies and that differs from many polymerases, including the eukaryotic poly(A) polymerases. Residues in the active site of VP55, located between the catalytic and C-terminal domains, make specific interactions with the adenine of the ATP analog, establishing the molecular basis of ATP recognition. VP55's concave surface docks the globular VP39. A model for RNA primer binding that involves all three VP55 domains and VP39 is proposed. The model supports biochemical evidence that VP39 functions as a processivity factor by partially enclosing the RNA primer at the heterodimer interface.     

RNA binding characteristics and overall topology of the vaccinia poly(A) polymerase-processivity factor-primer complex.

The vaccinia virus-encoded heterodimer responsible for poly(A) tail elongation comprises a polyadenylylation catalytic subunit (VP55) and associated processivity factor (VP39). We show that monomeric VP39's affinity for RNA homopolymers follows the hierarchy poly(I) >poly(U) >poly(G) >poly(A) >poly(C), that the heterodimer interacts stably with 40-45 nucleotide nucleic acid segments, and that its homopolymer preference for polyadenylylation priming is comparable to the VP39 affinity hierarchy (above). For oligonucleotide ligands possessing the previously-identified (rU)2-(N)25-rU heterodimer-binding motif, the heterodimer's affinity and base-type preference are mediated via both the (rU)2and rU portions, with the greater contribution coming from (rU)2. VP39's R107 sidechain contributes to specificity at the downstream rU. Substitution of each ribouridylate of the motif with either ribothymidine or 4-thiodeoxythymidine indicated that the downstream rU interacts with both heterodimer subunits, whereas the upstream (rU)2interacts only with VP55. A 'crosslinking SELEX' approach indicated VP39-base proximity around position -10 of a 4-thioribouridine/deoxycytidine ligand pool. Upon incubating the heterodimer with a panel of identical-sequence oligonucleotides derivatized with azidophenacyl bromide at various phosphate positions, those derivatized at positions -11 to -21 photocrosslinked to both subunits in a coordinated manner. This region may therefore pass through a 'cleft' or enclosed 'channel' at the subunit interface.     

Molecular flexibility and discontinuous translocation of a non-templated polymerase

Little is known regarding the translocation of non-templated nucleic acid polymerases with respect to single-stranded primers. VP55, the vaccinia virus poly(A) polymerase, translocates as it processively adds a approximately 3-7 adenylate tail to primers possessing only three ribouridylate residues (as an (rU)(2)-N(15)-rU motif), and a approximately 25-30 adenylate tail to primers that are more U-rich. Here, three models were addressed for the translocation of VP55 with respect to its primer, namely: (a) rigid protein/rigid nucleic acid; (b) flexible protein/rigid nucleic acid; (c) rigid protein/flexible nucleic acid. Analysis of free and covalently VP55-attached primers favored either (b) or a version of (c) incorporating a passive steric block, and suggested two regions of relative motion between polymerase and primer. Inclusion of a 6nt uridylate-rich patch at the primer 3' end switched the polymerase from approximately 3-7 nt to approximately 25-30 nt tail addition without affecting initial binding affinity. By synthesizing this patch as a (rU/dC) pool, discontinuous polymerase movements could be detected.     

Uridylate-containing RNA sequences determine specificity for binding and polyadenylation by the catalytic subunit of vaccinia virus poly(A) polymerase.

VP55, the catalytic subunit of vaccinia virus poly(A) polymerase, has the remarkable property of adding 30-35 adenylates to RNA 3' ends in a rapid processive burst before an abrupt transition to slow, non-processive adenylate addition. Here, we demonstrate that this property results from the affinity of the enzyme for uridylate residues within the 3' 31-40 nt of the RNA primer. At physiological salt concentrations, both polyadenylation and stable VP55 binding required the presence of multiple uridylates within a 31-40 nt length of RNA, though specific RNA sequences were not necessary. Even DNA in which the deoxythymidylate residues were replaced with ribouridylates, could be polyadenylated in a processive manner. Both the unmethylated pyrimidine ring and a 2'-OH on the associated sugar are features of ribouridylates that are important for priming. The abrupt termination of processive polyadenylation was attributed to translocation of VP55 along the nascent poly(A) tail, which lacks uridylates for stable binding. As evidence for translocation and interaction with newly synthesized RNA, other homopolymer tails were synthesized by VP55 in the presence of Mn2+, which relaxes its donor nucleotide specificity. Only during poly(U) tail synthesis did processive nucleotide addition fail to terminate.     

Molecular and biological characterization of deformed wing virus of honeybees (Apis mellifera L.)

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5' nontranslated leader sequence and a 317-nucleotide 3' nontranslated region, followed by a poly(A) tail. The three major structural proteins, VP1 (44 kDa), VP2 (32 kDa), and VP3 (28 kDa), were identified, and their genes were mapped to the N-terminal section of the polyprotein. The C-terminal part of the polyprotein contains sequence motifs typical of well-characterized picornavirus nonstructural proteins: an RNA helicase, a chymotrypsin-like 3C protease, and an RNA-dependent RNA polymerase. The genome organization, capsid morphology, and sequence comparison data indicate that DWV is a member of the recently established genus Iflavirus.     

Saltatory forward movement of a poly(A) polymerase during poly(A) tail addition

Vaccinia poly(A) polymerase (VP55) interacts with > or = 33-nucleotide (nt) primers via uridylates at two sites (-27/-26 and -10). It adds approximately 30-nt poly(A) tails with a rapid, processive burst in which the first few nt are added without substantial primer movement, and addition of the remaining adenylates is dependent upon a six-uridylate tract at the extreme 3' end of the primer and accompanied by polymerase translocation. Interaction of VP55 with 2-aminopurine (2-AP)-containing primers was associated with a 3-fold enhancement in 2-AP fluorescence. In stopped-flow experiments, fluorescence intensity changed with time during the polyadenylation burst in a manner dependent upon the position of 2-AP, indicating a non-uniform isomerization of the polymerase-primer complex with time consistent with a discontinuous (saltatory) translocation mechanism. Three distinct translocatory phases could be discerned: a -10(U)-binding site forward movement, a -27/-26(UU)-binding site jump to -10, then a -27/-26(UU)-binding site movement further downstream. Poly(A) tail elongation showed no apparent pauses during these isomerizations. Fluorescence changes during polyadenylation of 2-AP-containing primers with short preformed oligo(A) tails reinforced the above observations. Primers composed entirely of oligo(U) (apart from the 2-AP sensor), in which the polymerase modules might be most able to "slide" uniformly, also showed the characteristic saltatory pattern of translocation. These data indicate, for the first time, a discontinuous mode of translocation for a non-templated polymerase.     

Structurally and immunologically distinct poly(A) polymerases in rat liver. Occurrence of a tumor-type enzyme in normal liver

Poly(A) polymerases purified from rat liver nuclei consisted of two distinct species, a predominant enzyme of Mr = 38,000 and a minor one of Mr = 48,000. Prior to extensive purification, the minor enzyme constituted approximately 1% of the total liver poly(A) polymerase. Poly(A) polymerase purified from a rat tumor, Morris hepatoma 3924A, was comprised of a single species of Mr = 48,000 which was identical to the minor liver enzyme with respect to chromatographic and immunological characteristics. Gel filtration on Sephacryl S-200 using 0.3 M NaCl for elution showed that the major liver poly(A) polymerase had a molecular weight of 156,000, which corresponded to a tetramer of the 38-kDa polypeptide, whereas the hepatoma and minor liver 48-kDa species existed as dimers with a molecular weight of 96,000. Fractionation by Sephacryl S-200 resulted in complete loss of both liver poly(A) polymerase activities which could be restored by exogenous N1-type protein kinase. Following CNBr cleavage, the 48-kDa poly(A) polymerase from liver and hepatoma exhibited nearly identical peptide maps which were distinct from that of the major liver enzyme (38 kDa). Antibodies raised against tumor poly(A) polymerase reacted with the 48-kDa polypeptide but not with the 38-kDa liver enzyme. Immune complex formation was observed between seven of the eight CNBr cleavage products derived from the 48-kDa polypeptide of both liver and hepatoma. It is concluded that distinct genes in rat liver code for two structurally and immunologically unique nuclear poly(A) polymerases, one of which is identical to the enzyme from the hepatoma.     

The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance

Human DNA polymerase gamma is composed of a 140-kDa catalytic subunit and a smaller accessory protein variously reported to be 43-54 kDa. Immunoblot analysis of the purified, heterodimeric native human polymerase gamma complex identified the accessory subunit as 55 kDa. We isolated the full-length cDNA encoding a 55-kDa polypeptide, expressed the cDNA in Escherichia coli and purified the 55-kDa protein to homogeneity. Recombinant Hp55 forms a high affinity, salt-stable complex with Hp140 during protein affinity chromatography. Immunoprecipitation, gel filtration, and sedimentation analyses revealed a 190-kDa complex indicative of a native heterodimer. Reconstitution of Hp140.Hp55 raises the salt optimum of Hp140, stimulates the polymerase and exonuclease activities, and increases the processivity of the enzyme by several 100-fold. Similar to Hp140, isolated Hp55 binds DNA with moderate strength and was a specificity for double-stranded primer-template DNA. However, Hp140.Hp55 has a surprisingly high affinity for DNA, and kinetic analyses indicate Hp55 enhances the affinity of Hp140 for primer termini by 2 orders of magnitude. Thus the enhanced DNA binding caused by Hp55 is the basis for the salt tolerance and high processivity characteristic of DNA polymerase gamma. Observation of native DNA polymerase gamma both as an Hp140 monomer and as a heterodimer with Hp55 supports the notion that the two forms act in mitochondrial DNA repair and replication. Additionally, association of Hp55 with Hp140 protects the polymerase from inhibition by N-ethylmaleimide.     

Identification of new poly(A) polymerase-inhibitory proteins capable of regulating pre-mRNA polyadenylation

The 3' ends of nearly all eukaryotic pre-mRNAs undergo cleavage and polyadenylation, thereby acquiring a poly(A) tail added by the enzyme poly(A) polymerase (PAP). Two well-characterized examples of regulated poly(A) tail addition in the nucleus consist of spliceosomal proteins, either the U1A or U170K proteins, binding to the pre-mRNA and inhibiting PAP via their PAP regulatory domains (PRDs). These two proteins are the only known examples of this type of gene regulation. On the basis of sequence comparisons, it was predicted that many other proteins, including some members of the SR family of splicing proteins, contain functional PRDs. Here we demonstrate that the putative PRDs found in the SR domains of the SR proteins SRP75 and U2AF65, via fusion to a heterologous MS2 RNA binding protein, specifically and efficiently inhibit PAP in vitro and pre-mRNA polyadenylation in vitro and in vivo. A similar region from the SR domain of SRP40 does not exhibit these activities, indicating that this is not a general property of SR domains. We find that the polyadenylation- and PAP-inhibitory activity of a given polypeptide can be accurately predicted based on sequence similarity to known PRDs and can be measured even if the polypeptides' RNA target is unknown. Our results also indicate that PRDs function as part of a network of interactions within the pre-mRNA processing complex and suggest that this type of regulation will be more widespread than previously thought.     

Kinetic proofreading scanning models for eukaryotic translational initiation: the cap and poly(A) tail dependency of translation

Two simplified kinetic proofreading scanning (KPS) models were proposed to describe the 5' cap and 3' poly(A) tail dependency of eukaryotic translation initiation. In Model I, the initiation factor complex starts scanning and unwinding the secondary structure of the 5' untranslated region (UTR) from the 5' terminus of mRNA. In Model II, the initiation factor complex starts scanning from any binding site in the 5' UTR. In both models, following ATP hydrolysis, the initiation factor complex either dissociates from mRNA or continues to scan and unwind RNA secondary structure in the 5' UTR. This step repeats n times until the AUG codon is reached. These two models show very different cap and/or poly(A) tail dependency of translation initiation. The models predict that both cap and poly(A) tail dependencies of translation, and translatability of mRNAs are coupled with the structure of 5' UTR: the translation of mRNA with structured 5' UTR is strongly cap- and poly(A) tail-dependent; while translation of mRNA with unstructured 5' UTR is less cap- and poly(A) tail-dependent. We use these two models to explain: (1) the cap and poly(A) tail dependence of translation; (2) the effect of exogenous poly(A) on translation; (3) repression of host mRNA and translation of late adenovirus mRNA in the late phase of adenovirus infection; (4) repression of host mRNA and translation of Vaccinia virus mRNA in virus-infected cell; (5) heat shock repression of translation of normal mRNA and stimulation of translation of hsp mRNA; and (6) the synergistic effect of cap and poly(A) tail on stimulating translation. The kinetic proofreading scanning models provide a coherent interpretation of those phenomena.     

Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends.

VP39 is a bifunctional vaccinia virus protein that acts as both a cap- dependent 2'-O-Methyltransferase and a poly(A) polymerase processivity factor. An analysis of C-terminal truncation mutants of a GST-VP39 fusion protein indicated the presence of a protease-sensitive C-terminal "tail" 36-43 amino acids in length that is non-essential for VP39 function. Fourteen new VP39 pointmutants, containing either single or multiple-clustered amino acid substitutions, were expressed in Escherichia coli. Of the eight that retained either one or both of the activities of VP39, seven were specifically methyltransferase-defective. None was specifically defective in adenylyltransferase stimulation. The nature of the methyltransferase defects in 10 of the methyltransferase-specific defectives, identified both herein and in a previous study (Schnierle BS, Gershon PD, Moss B, 1994, J biol Chem 269:20700-20706), was investigated using two novel substrate-binding assays. Three of the mutants (and possible a fourth), whose lesions were juxtaposed and centrally located within VP39, exhibited anomalous S-adenosyl-(L)-methionine (AdoMet) binding behavior, identifying residues important for AdoMet binding and possible also for catalysis. A surface plasmon resonance-based assay measured the interaction of VP39 with uncapped and 5'-cap 0-terminated oligo(A). A cap 0- dependent association-rate enhancement was observed for wild-type VP39 and 4 of the 10 mutant proteins. Two others were identified as defective in cap binding, and a third as partially defective. The lesions within the latter three mutants were closely apposed, and located toward the N-terminus of VP39. We have thus identified regions of VP39 important for interaction with its two substrates for cap-dependent methyltransferase activity: AdoMet and cap 0.     

Structure and function of poly(A) binding proteins

Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.     

The synthesis of minus-strand RNA of bamboo mosaic potexvirus initiates from multiple sites within the poly(A) tail

The 3' terminus of the bamboo mosaic potexvirus (BaMV) contains a poly(A) tail, the 5' portion of which participates in the formation of an RNA pseudoknot required for BaMV RNA replication. Recombinant RNA-dependent RNA polymerase (RdRp) of BaMV binds to the pseudoknot poly(A) tail in gel mobility shift assays (C.-Y. Huang, Y.-L. Huang, M. Meng, Y.-H. Hsu, and C.-H. Tsai, J. Virol. 75:2818-2824, 2001). Approximately 20 nucleotides of the poly(A) tail adjacent to the 3' untranslated region (UTR) are protected from diethylpyrocarbonate modification, suggesting that this region may be used to initiate minus-strand RNA synthesis. The 5' terminus of the minus-strand RNA synthesized by the RdRp in vitro was examined using 5' rapid amplification of cDNA ends (RACE) and DNA sequencing. Minus-strand RNA synthesis was found to initiate from several positions within the poly(A) tail, with the highest frequency of initiation being from the 7th to the 10th adenylates counted from the 5'-most adenylate of the poly(A) tail. Sequence analyses of BaMV progeny RNAs recovered from Nicotiana benthamiana protoplasts which were inoculated with mutants containing a mutation at the 1st, 4th, 7th, or 16th position of the poly(A) tail suggested the existence of variable initiation sites, similar to those found in 5' RACE experiments. We deduce that the initiation site for minus-strand RNA synthesis is not fixed at one position but resides opposite one of the 15 adenylates of the poly(A) tail immediately downstream of the 3' UTR of BaMV genomic RNA.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.