In vitro studies of the Prp9.Prp11.Prp21 complex indicate a pathway for U2 small nuclear ribonucleoprotein activation.

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9.Prp11.Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9.Prp11.Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide- directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.

Transcriptional pausing, arrest, and readthrough at the adenovirus major late attenuation site.

RNA polymerase II (pol II) transcription complexes initiated from the adenovirus major late promoter can become blocked both in vitro and in vivo at a specific site within the first intron of the transcription unit. In vitro, polymerases that fail to read through the major late attenuation site remain stably bound to the template in a ternary complex that is indefinitely blocked from continuing elongation, a phenomenon referred to as "arrest." Elongation factor SII has been shown both to promote readthrough of this and other arrest sites and to stimulate a previously unknown 3' to 5' exonuclease activity of pol II. We have proposed that the two activities are related and that SII promotes readthrough by means of the enhancement of the exonuclease activity. In the experiments reported here, we have tested several features of that model. In particular, we have examined the hypothesis that SII stimulates readthrough by allowing the polymerase to undergo multiple cycles of removal and resynthesis of RNA bases preceding the attenuation site. In addition, we present experimental support for the proposal that the length of time polymerase pauses at the attenuation site is important to the efficiency of arrest. The results of these experiments are discussed in the context of the model.

Mechanistic studies of transcription arrest at the adenovirus major late attenuation site. Comparison of purified RNA polymerase II and washed elongation complexes.

Transcription elongation in a nuclear extract in vitro is efficiently blocked by Sarkosyl at a specific site downstream of the adenovirus major late (ML) promoter at which regulated transcription arrest has also been observed in vivo. In the experiments reported here, we examined the response of the polymerase to the ML attenuation site in two assay systems: 1) purified RNA polymerase II (pol II) transcribing tailed templates and 2) elongation complexes formed on immobilized templates and then depleted of elongation factors by extensive washing. Efficient site-specific arrest occurred in both systems, demonstrating that recognition of the site is an intrinsic property of the polymerase. However, the elongation properties of washed elongation complexes and purified pol II were not equivalent. In particular, the efficiency of arrest of washed elongation complexes was influenced both by the promoter from which transcription was initiated and by DNA sequences upstream from the attenuation site that did not contribute to the arrest of purified pol II. The polymerase and washed elongation complexes both remained in stable ternary complexes at the ML site with a lifetime of hours; addition of the elongation factor SII to these complexes promoted resumption of elongation. The efficiency of arrest in both systems was dependent on the solution concentration of the nucleotide incorporated at +187 (just beyond the attenuation site), indicating that pausing is an important part of the arrest mechanism. Based on this and other findings, we argue that the polymerase assumes an altered, elongation-incompetent conformation when arrest occurs.

In vitro analysis of a transcription termination site for RNA polymerase II.

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.