Enhanced binding to origin DNA at low pH enables easy detection of polyomavirus large T antigen by gel mobility shift assay of unfixed complexes.

Enhanced, stable binding by polyomavirus large T antigen to the viral DNA replication origin at pH 6 allowed the development of a gel mobility shift assay for the detection of large T antigen. Such assays were not possible at pH 7.6 without previous fixation, due to instability of the complexes. We demonstrated that the gel mobility shift assay at pH 6 is very sensitive, allowing the detection of as little as 5 ng large T antigen, and is highly specific for DNA containing G(A/G)GGC target sequences. This method was used to detect large T antigen in crude cell lysates from transformed yeast cell lines or nuclear extracts from infected insect cells. Large T antigen-DNA complexes remained at or near the loading well in 5% acrylamide or 1.5% agarose gels, indicating that these complexes are very large. Glycerol gradient analysis showed that protein-DNA complexes formed at pH 6 were massive, and that large T antigen also formed large complexes when incubated at low pH in the absence of DNA. These results show that pH has a major effect on binding of large T antigen to its multiple target sites in the viral origin of DNA replication, presumably by affecting protein-protein interactions that are important for the stability of large T antigen-DNA complexes.

Polyomavirus large T antigen binds cooperatively to its multiple binding sites in the viral origin of DNA replication.

Polyomavirus large T antigen binds to multiple 5'-G(A/G)GGC-3' pentanucleotide sequences in sites 1/2, A, B, and C within and adjacent to the origin of viral DNA replication on the polyomavirus genome. We asked whether the binding of large T antigen to one of these sites could influence binding to other sites. We discovered that binding to origin DNA is substantially stronger at pH 6 to 7 than at pH 7.4 to 7.8, a range often used in DNA binding assays. Large T antigen-DNA complexes formed at pH 6 to 7 were stable, but a fraction of these complexes dissociated at pH 7.6 and above upon dilution or during electrophoresis. Increased binding at low pH is therefore due at least in part to increased stability of protein-DNA complexes, and binding at higher pH values is reversible. Binding to fragments of origin DNA in which one or more sites were deleted or inactivated by point mutations was measured by nitrocellulose filter binding and DNase I footprinting. The results showed that large T antigen binds cooperatively to its four binding sites in viral DNA, suggesting that the binding of this protein to one of these sites stabilizes its binding to other sites via protein-protein contacts. Sites A, B, and C may therefore augment DNA replication by facilitating the binding of large T antigen to site 1/2 at the replication origin. ATP stabilized large T antigen-DNA complexes against dissociation in the presence, but not the absence, of site 1/2, and ATP specifically enhanced protection against DNase I digestion in the central 10 to 12 bp of site 1/2, at which hexamers are believed to form and begin unwinding DNA. We propose that large T antigen molecules bound to these multiple sites on origin DNA interact with each other to form a compact protein-DNA complex and, furthermore, that ATP stimulates their assembly into hexamers at site 1/2 by a "handover" mechanism mediated by these protein-protein contacts.

A conserved cysteine residue in yeast uroporphyrinogen decarboxylase is not essential for enzymatic activity.

Uroporphyrinogen decarboxylase catalyzes the fifth step of heme biosynthesis in Saccharomyces cerevisiae. Studies utilizing sulfhydryl-specific reagents suggest that the enzyme requires a cysteine residue within the catalytic site. This hypothesis was tested directly by site-directed mutagenesis of highly conserved cysteine-52 to serine or alanine. Plasmids containing these mutations were able to complement a hem6 mutant strain. In addition, properties associated with decreased uroporphyrinogen decarboxylase activity were not detected in the mutant strain transformed with these mutant plasmids. These results suggest that the conserved cysteine-52 by itself is not essential for enzymatic activity.

Production of active polyomavirus large T antigen in yeast Pichia pastoris.

The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immunoaffinity chromatography. We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.

Novel exonic elements that modulate splicing of the human fibronectin EDA exon.

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3'-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric beta-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.

The HMG domain of the ROX1 protein mediates repression of HEM13 through overlapping DNA binding and oligomerization functions.

The ROX1 gene of Saccharomyces cerevisiae encodes a protein required for the repression of genes expressed under anaerobic conditions. ROX1 belongs to a family of DNA binding proteins which contain the high mobility group motif (HMG domain). To ascertain whether the HMG domain of ROX1 is required for specific DNA binding we synthesized a series of ROX1 protein derivatives, either in vitro or in Escherichia coli as fusions to glutathione S-transferase (GST) protein, and tested them for their ability to bind to DNA. Both ROX1 proteins that were synthesized in vitro and GST-ROX1 fusion proteins containing the intact HMG domain were able to bind to specific target DNA sequences. In contrast, ROX1 proteins which contained deletions within the HMG domain were no longer capable of binding to DNA. The oligomerization of ROX1 in vitro was demonstrated using affinity-purified GST-ROXI protein and ROX1 labelled with [35S]methionine. Using various ROX1 protein derivatives we were able to demonstrate that the domain required for ROX1-ROX1 interaction resides within the N-terminal 100 amino acids which constitute the HMG domain. Therefore, the HMG domain is required for both DNA binding activity and oligomerization of ROX1.

RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA.

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.

Mutation of large T-antigen-binding site A, but not site B or C, eliminates stalling by RNA polymerase II in the intergenic region of polyomavirus DNA.

During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus.

Stalling by RNA polymerase II in the polyomavirus intergenic region is dependent on functional large T antigen.

RNA polymerase II encounters an elongation block and stalls in vivo during transcription of the late strand of polyomavirus DNA. In this study, we performed transcriptional run-on assays and localized the stalling site to a 164-nucleotide region (nt 11-175) that contains specific binding sites for polyomavirus large T antigen. The effect of large T antigen on elongation by RNA polymerase II through this region was examined in cells infected with a mutant polyomavirus (AT3-ts25E) which encodes a thermolabile large T antigen. Removal of functional large T antigen by shifting to the nonpermissive temperature (39 degrees) eliminated stalling by RNA polymerase in this region, although RNA polymerases transcribing other regions of the viral genome were unaffected. RNA polymerase resumed stalling when functional large T antigen was again allowed to accumulate by shifting back to the permissive temperature (32 degrees). We conclude that stalling by RNA polymerase II in vivo is dependent on the presence of functional large T antigen.

Determination of the origin-specific DNA-binding domain of polyomavirus large T antigen.

To map the DNA-binding domain of polyomavirus large T antigen, we constructed a set of plasmids coding for unidirectional carboxy- or amino-terminal deletion mutations in the large T antigen. Analysis of origin-specific DNA binding by mutant proteins expressed in Cos-1 cells revealed that the C-terminal boundary of the DNA-binding domain is at or near Glu-398. Fusion proteins of large T antigen lacking the first 200 N-terminal amino acids bound specifically to polyomavirus origin DNA; however, deletions beyond this site resulted in unstable proteins which could not be tested for DNA binding. Testing of point mutants and internal deletions by others suggested that the N-terminal boundary of the DNA-binding domain lies between amino acids 282 and 286. Taken together, these results locate the DNA-binding domain of polyomavirus large T antigen to the 116-amino-acid region between residues 282 and 398.

Production of polyomavirus late mRNAs requires sequences near the 5' end of the leader but does not require leader-to-leader splicing.

Polyomavirus late mRNAs contain multiple copies of a 57-nucleotide leader sequence derived by splicing from multigenome-length late transcripts. Inefficient termination of transcription and inefficient polyadenylation allow accumulation of these giant transcripts. In this report, we show that a viable mutant virus, ins5, which contains an efficient rabbit beta-globin polyadenylation signal, produced late mRNAs whose vast majority contains only one leader. ins5 virus nevertheless produced as much late mRNA as did wild-type virus and grew as well as did wild-type virus in mouse cells. These results demonstrate that leader-to-leader splicing per se is not required for efficient production of late mRNAs or for efficient virus replication. However, we also found that RNAs lacking critical sequences near the 5' end of the leader did not accumulate as mRNAs and that most late transcripts made during the early part of the late phase, when few late mRNAs are produced, initiated downstream of the 5' end of the leader. These results indicate that a sequence element near the 5' end of the leader is required for proper processing, transport, or stability of late mRNAs and that the control of late mRNA production depends in part on the choice of transcription initiation sites at the late promoter.

RNA polymerase II pauses in vitro, but does not terminate, at discrete sites in promoter-proximal regions on polyomavirus transcription complexes.

Many RNA polymerases stall and/or prematurely terminate transcription nearby the early and late promoters of polyomavirus in vivo during the late phase of productive infection. In this paper we analyzed the RNAs made when these promoter-proximal RNA polymerases were allowed to elongate their nascent chains in vitro on viral transcription complexes isolated from infected cells. RNA was labeled in the presence of a high specific activity of one [alpha-32P]-ribonucleoside triphosphate (rNTP) (less than 1 microM final concentration) and saturating concentrations of the other three rNTPs. Under these conditions, promoter-proximal RNAs of discrete sizes were produced. We show that these discrete RNA species are produced by pausing of RNA polymerase II due to limiting concentrations of one of the rNTPs, and that the positions of the pause sites depend on which rNTP is limiting. This pausing does not result in release of the RNA polymerase or the nascent RNA chain from the transcription complex, as these chains can be further extended when high concentrations of all four rNTPs are supplied. Our results conflict with the interpretations of other investigators who suggest that formation of discrete RNA products, under similar in vitro conditions, reflects authentic termination by RNA polymerase II.

RNA polymerases stall and/or prematurely terminate nearby both early and late promoters on polyomavirus DNA.

Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.

A rabbit beta-globin polyadenylation signal directs efficient termination of transcription of polyomavirus DNA.

We constructed a viable insertion mutant (ins 5) of polyomavirus which contains, upstream of the L-strand polyadenylation signal, a 94-nt fragment of rabbit beta-globin DNA. Included in this fragment are all of the sequence elements required for efficient cleavage and polyadenylation of rabbit beta-globin RNA. The beta-globin signal was efficiently recognized by the cleavage/polyadenylation machinery in mouse 3T6 cells infected with ins 5, signalling greater than 90% of the polyadenylation events on L-strand RNAs. Furthermore, the presence of this efficient polyadenylation signal resulted in a 1.4- to 2.5-fold increase in the fraction of virus-specific RNAs that were polyadenylated. Most importantly, termination of transcription by RNA polymerase II on ins 5 DNA was also increased compared with wild-type virus; nearly 100% of polymerases terminated per traverse of the ins 5 genome. These findings demonstrate that the rabbit beta-globin insert, which contains a strong polyadenylation signal, also contains at least part of a signal for termination of transcription by RNA polymerase II. These results also show that the multiple, spliced leaders on polyomavirus L-strand mRNAs, which arise as a result of inefficient termination and polyadenylation, are not necessary for efficient virus replication.

Duplication of functional polyadenylation signals in polyomavirus DNA does not alter efficiency of polyadenylation or transcription termination.

We constructed viable insertion mutants of polyomavirus that contain duplications of the nucleotide sequences surrounding the polyadenylation sites for both E- and L-strand RNAs. Our results showed that formation of poly(A)+ 3'termini of L-strand mRNAs requires sequence elements located between 12 and 87 nucleotides downstream of AAUAAA. No more than 19 nucleotides upstream and 44 nucleotides downstream of AAUAAA are required for polyadenylation of E-strand mRNAs. Our results and those of others suggest that there are three distinct sequence elements required for mRNA 3' end formation: AAUAAA and two downstream elements. An insertion mutant containing two adjacent functional polyadenylation signals produced E-strand and L-strand mRNAs with 3' ends at both sites. However, the overall level of polyadenylation of L-strand RNAs was not increased over the low (10 to 25%) levels seen with wild-type virus. Neither was the efficiency of termination of L-strand transcription increased in mutant virus-infected cells. We conclude that factors required for both polyadenylation and transcription termination are limiting in polyomavirus-infected mouse cells.

Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

We devised a strategy to measure the efficiency of transcription termination in vivo by RNA polymerase on polyomavirus DNA. Pulse-labeled nuclear RNA was hybridized with a single-stranded polyomavirus DNA fragment which spans the transcription initiation region. Hybrids were treated with RNase, bound to nitrocellulose filters, eluted with S1 nuclease, and analyzed by gel electrophoresis. The ratio of full-length to less-than-full-length DNA-RNA hybrids was used to calculate transcription termination frequency. We found that 50% of the polymerases terminated per traverse of the L DNA strand during the late phase of infection. The method for mapping in vivo pulse-labeled RNAs which we developed is potentially useful for studying unstable cellular or viral RNAs.

Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

The rate and efficiency of polyadenylation of late polyomavirus RNA in the nucleus of productively infected mouse kidney cells were determined by measuring incorporation of [3H]uridine into total and polyadenylated viral RNAs fractionated by oligodeoxythymidylic acid-cellulose chromatography. Polyadenylation is rapid: the average delay between synthesis and polyadenylation of viral RNA in the nucleus is 1 to 2 min. However, only 10 to 25% of viral RNA molecules become polyadenylated. Polyadenylated RNAs in the nucleus are a family of molecules which differ in size by an integral number of viral genome lengths (5.3 kilobases). These RNAs are generated by repeated passage of RNA polymerase around the circular viral DNA, accompanied by addition of polyadenylic acid to a unique 3' end situated 2.2 + n(5.3) kilobases from the 5' end of the RNAs (n can be an integer from 0 to at least 3). Between 30 and 50% of the sequences in nuclear polyadenylated RNA are conserved during processing and transport to the cytoplasm as mRNA. This is consistent with the molar ratios of nuclear polyadenylated RNAs in the different size classes, and it suggests that most polyadenylated nuclear RNA is efficiently processed to mRNA. Thus, the low overall conservation of viral RNA sequences between nucleus and cytoplasm is explained by (i) low efficiency of polyadenylation of nuclear RNA and (ii) removal of substantial parts of polyadenylated RNAs during splicing. The correlation between inefficient termination of transcription and inefficient polyadenylation of transcripts suggests that these two events may be causally linked.

Synthesis of prematurely terminated late transcripts of polyoma virus DNA is resistant to inhibition by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Exposure of mouse kidney cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (75 to 300 microM) during the late phase of infection by polyoma virus resulted in nearly complete (90 to 98%) inhibition of virus RNA synthesis. Sedimentation analysis revealed that, although the synthesis of high mol. wt. (greater than 10S) virus RNA was inhibited in a manner parallel to that of total virus RNA, the synthesis of small (3S to 7S) virus RNA was inhibited by only 40 to 50% in the presence of DRB. As a result, virus RNA synthesized in the presence of DRB contained a peak at 3S to 7S in addition ot residual high mol. wt. virus RNA. Small virus RNA from either untreated or DRB-treated cells contained three- to sixfold higher levels of transcripts from the DNA fragment which lies between the BamHI and Bg/I sites (58 . 0 to 72 . 2 map units) than from DNA fragments covering the rest of the virus genome. Furthermore, 80% of the small RNA which hybridized to this fragment was complementary to the L strand of virus DNA. These results suggest that L strand transcripts are initiated within the Bg/I-BamHI DNA fragment and that a portion of these transcripts is prematurely terminated within several hundred nucleotides of the site(s) of initiation. DRB had little effect on the synthesis of these prematurely terminated RNAs.

Efficiency of processing of viral RNA during the early and late phases of productive infection by polyoma virus.

The efficiency of processing of polyoma viral RNA and of its export from nucleus to cytoplasm was measured in primary mouse kidney cells by comparing the initial rates of incorporation of [3H]uridine into cytoplasmic and nuclear viral RNA. Appropriate methods of cell fractionation were chosen to maximize yields of cytoplasmic RNA and to minimize leakage of nuclear RNA. Incorporation of [3H]uridine into cellular 4S RNA in the cytoplasm was followed to monitor pool equilibration and maintenance of an excess of radioactive precursor throughout the experimental period. During the early phase of infection (9 to 11 h, in the presence of 5-fluorodeoxyuridine), viral RNA was rapidly and efficiently exported from nucleus to cytoplasm. Viral RNA appeared in the cytoplasm within 6 min of its synthesis, greater than half of the viral RNA synthesized in the nucleus was exported to the cytoplasm. In contrast, during the late phase of infection (28 to 30 h), viral RNA was exported more slowly, appearing in the cytoplasm 12 to 20 min after its synthesis, and much less efficiently-only 5% of late nuclear transcripts was exported. The poor efficiency of processing of late viral RNA may be, in part, a result of (i) the presence in nuclear transcripts of non-mRNA sequences which are removed during processing; (ii) the presence in nuclear transcripts of multiple copies of mRNA sequences, only one of which is incorporated into mature mRNA; and (iii) inefficient polyadenylation of viral nuclear RNA.

Extent of transcription of the E strand of polyoma virus DNA during the early phase of productive infection.

Early polyoma virus-specific RNA, in nuclei and cytoplasm of cells labeled with [(3)H]uridine, was analyzed by hybridization with filter-bound Hpa II fragments of polyoma DNA. About 40% of labeled cytoplasmic virus-specific RNA hybridized with Hpa II fragment 2, which represents about 40% of the region coding for E-strand mRNA's; less than 5% hybridized with fragments 1 or 3, which lie outside this region. A somewhat lower proportion (about 30%) of labeled nuclear virus-specific RNA hybridized with fragment 2, and a small but significant fraction (7 to 14%) hybridized with fragments 1 and 3. About two-thirds of the nuclear RNA which hybridized to fragment 1 was complementary to the E strand, and one-third was complementary to the L strand. Results did not vary greatly in samples labeled for periods of from 15 min to 3 h. The major species of pulse-labeled nuclear polyoma-specific RNA sedimented at 22S and thus is slightly larger than the 19S cytoplasmic mRNA. These results show that most early nuclear RNA ( approximately 75%) is transcribed from the region of the E strand, which codes for early mRNA's, and that there is probably a site at which transcription is terminated at the end of this region. However, a small amount of early nuclear RNA ( approximately 15%) is transcribed from the remainder of the E strand, perhaps by readthrough of this termination signal. In addition, there is a small amount of transcription from the L strand, whose significance is unclear. Neither the L-strand transcripts nor the nonmessenger E-strand transcripts are transported to the cytoplasm.