Sedimentation analysis of polyadenylation-specific complexes.

Precursor RNA containing the adenovirus L3 polyadenylation site is assembled into a 50S complex upon incubation with HeLa nuclear extract at 30 degrees C. The cofactor and sequence requirements for 50S complex formation are similar to those of the in vitro polyadenylation reaction. Assembly of this complex requires ATP but is not dependent upon synthesis of a poly(A) tract. In addition, a 50S complex does not form on substrate RNA in which the AAUAAA hexanucleotide upstream of the poly(A) site has been mutated to AAGAAA or on RNA in which sequences between +5 and +48 nucleotides downstream of the site have been removed. These mutations also prevent in vitro processing of substrate RNA. Kinetic studies suggest that the 50S complex is an intermediate in the polyadenylation reaction. It forms at an early stage in the reaction and at later times contains both poly(A)+ RNA as well as unreacted precursor. U-type small nuclear ribonucleoprotein particles are components of the 50S complex, as shown by immunoprecipitation with antiserum specific to the trimethyl cap of these small nuclear RNAs.

Transcription termination in animal viruses and cells.

Three experimental systems: isolated nuclei, cell-free reactions and whole cells were used for defining and characterizing cis and trans elements which regulate the block of transcription elongation in animal viruses and cells. In addition we have presented models for transcription termination within and at the end of a gene, which are consistent with the available information on the transcription bubble propagated during transcription elongation and can explain the modes of transcription termination described for various eukaryotic genes.

Pausing of RNA polymerase molecules during in vivo transcription of the SV40 leader region.

Viral transcription complexes were isolated from SV40-infected cells and incubated in vitro in the presence of [alpha-32P]UTP to allow elongation of the promoter-proximal RNA up to the attenuation sites. The 94 nucleotide attenuated RNA (spanning nucleotides 243-336) was purified, digested with RNase T1 and fingerprinted. The labeled oligonucleotides were then isolated, digested with RNase T2 and their base composition was determined. Based on these analyses 10 consecutive oligonucleotides, spanning residues 259-336, were identified. As the in vivo synthesized oligonucleotides are unlabeled the junctions between labeled and unlabeled oligonucleotides define the in vivo pause sites of RNA polymerase molecules. The characterization of the 10 radioactive spots and their relative intensities allowed the localization of two in vivo pause sites: one at 13-16 nucleotides downstream from the major initiation site presumably at the initial opening of the DNA helix and the second at approximately 40 nucleotides downstream from the major initiation site, just past a GC-rich region of dyad symmetry. It is postulated that pausing of RNA polymerase molecules in the leader region is an essential process in the control of SV40 late transcription.

Analysis of RNA cleavage at the adenovirus-2 L3 polyadenylation site.

Processing at the L3 polyadenylation site of human adenovirus-2 involves endonucleolytic cleavage generating the 3' terminal sequence -UAOH to which adenosine residues are added. This dinucleotide is 19 nucleotides downstream of the AAUAAA polyadenylation signal. The ATP analog cordycepin triphosphate (3' dATP) inhibits poly(A) synthesis, but precursor RNA is processed to give a product terminating in -UAAH. Addition of only one adenosine analog demonstrates that the initial poly(A) tract is synthesized by polymerization of single residues rather than by ligation of preformed poly(A). Cleavage is not coupled to polyadenylation since incubation with an ATP analog containing a non-hydrolyzable alpha--beta bond generates a product with a 3' terminus coincident with the -UAOH) addition site. Addition of this accurately processed RNA to a nuclear extract results in efficient polyadenylation, suggesting that downstream sequences are not required for synthesis of the poly(A) tract. Finally, processing at the L3 poly(A) site may involve both endonucleolytic and exonucleolytic activities.

Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.

Sedimentation analysis of pulse-labeled RNA synthesized in nuclei isolated from simian virus 40-infected cells revealed an abundance of short cellular and viral RNAs. The relative amount of the short chains is increased in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The short viral RNAs were purified by hybridization to and elution from simian virus 40 DNA on filters, and their sizes were determined by gel electrophoresis. A major band of 93- to 95-nucleotide-long RNA was observed along with additional minor bands. Identical bands were revealed when the viral RNA was purified from nuclei of cells pretreated with DRB. The major band was identified as an aborted transcript of a RNA that initiated at the major initiation site (nucleotide 243). We have found that the DNA region where the RNA stops is A+T rich and is immediately preceded by a G+C-rich region that exhibits dyad symmetry, resembling the termination signal in prokaryotes. These observations show that RNA polymerase II responds to the same termination signal as the prokaryotic enzyme and suggest that a mechanism of attenuation regulates simian virus 40 late transcription.

Attenuation in the control of SV40 gene expression.

Nuclei and viral transcriptional complexes were prepared from cells infected with simian virus 40 and incubated in vitro in the presence of alpha- 32P-UTP. The in vitro elongated viral RNA appeared with a peak of 5S in sucrose gradients and hybridized preferentially to a promoter-proximal region of SV40 DNA. Treatment of infected cells with proflavine led to transcription of elongated RNA, while treatment of cells with 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole, a drug known to enhance premature termination, augmented accumulation of the promoter-proximal RNA. The in vitro elongated RNA produced a major band of 93-95 nucleotides in length in acrylamide gel. This RNA was found to map between the major initiation site at nucleotide 243 and nucleotides 335-337. The significance of these observations with respect to the transcription termination signal and the control of SV40 gene expression is discussed.

Electrophoretic separation of polyadenylation-specific complexes.

A polyadenylation-specific complex composed of precursor RNA containing the adenovirus-2 L3 site and HeLa cellular components was detected by electrophoresis on a native, low-percentage polyacrylamide gel. Upon incubation in a reaction containing ATP and nuclear extract, precursor RNA was rapidly assembled into this complex. This assembly did not require poly(A) synthesis, as it occurred efficiently in the presence of ATP analogs that inhibited this reaction. Mutation of the hexanucleotide AAUAAA 20 nucleotides upstream of the L3 site to AAGAAA or deletion of sequence between +5 and +48 nucleotides downstream of the L3 site inactivates polyadenylation. The specific complex did not effectively from on substrate RNA with either the AAGAAA mutation or the downstream deletion mutation. Kinetic experiments showed that the assembly of this complex preceded processing of precursor RNA. We proposed that formation of this complex represents an intermediate step in polyadenylation.