The Saccharomyces cerevisiae protein Prp5 is a member of the "DEAD box" family of putative RNA-dependent ATPases and helicases. The protein was purified from Escherichia coli and determined to be an RNA-dependent ATPase. The ATPase activity is 7-fold more specific for full-length U2 than for any of the other small nuclear RNAs or nonspecific RNAs tested. An RNaseH assay in extracts was used to demonstrate that Prp5 mediates an ATP-dependent conformational change in the intact U2 small nuclear ribonucleoprotein. We propose that this conformational change makes the branch point pairing sequence of U2 RNA accessible for pairing with the intron allowing formation of the pre-spliceosome.Splicing of introns in nuclear pre-mRNA takes place in a complex structure, the spliceosome, which is assembled de novo for each splicing event. Once assembled, the spliceosome catalyzes two sequential trans-esterification reactions that remove the intron from the pre-mRNA and ligates the exons. In the first reaction, the 2Ј-OH of a particular adenosine residue near the 3Ј splice site attacks the phosphodiester bond at the 5Ј splice site giving the intermediates in the reaction, exon 1, and the lariat intron, exon 2, a branched structure in which the 5Ј-nucleotide is connected to the branch point adenosine via a 2Ј-5Ј phosphodiester bond. In the second reaction, the 3Ј-OH of exon 1 attacks the phosphodiester bond at the 3Ј splice site, resulting in the formation of the spliced mRNA product and releasing the intron in lariat form (1-3).The group II self-splicing introns are spliced by an identical pathway, leading to the principal tenets of this field that pre-mRNA splicing is an RNA-catalyzed reaction and that the two processes share a distant evolutionary ancestor (4).The spliceosome contains five ribonucleoprotein particles, snRNPs, 1 called U1, U2, U4, U5, and U6 and is assembled stepwise in a dynamic process in which the pre-mRNA and the snRNAs undergo a series of base pairing interactions resulting finally in the catalytically competent structure, presumably a structure sharing common chemical and catalytic features with the group II self-splicing introns. The process of spliceosome assembly is quite different from the other complex ribonucleoprotein machine with which it can be compared, the ribosome (5). The ribosome can self-assemble from pure RNA and protein components in vitro without the requirement for ATP hydrolysis or trans-acting proteins. The unique essence of pre-mRNA splicing is to be found in the ordered and precise assembly steps all but the first of which require ATP hydrolysis.Yeast genetic studies of proteins absolutely required for pre-mRNA splicing has thus far revealed only one source of the ATP requirement in spliceosome assembly. The proteins Prp2, Prp5, Prp16, Prp22, and Prp28 are all members of a superfamily defined by the first member of the family, the eukaryotic translation initiation factor eIF-4a (6 -11). These proteins share a number of sequence motifs including a characteristic ATP binding site whi...

No abstract

No abstract

We report the identification of a new gene, RRP3 (rRNA processing), which is required for pre-rRNA processing. Rrp3 is a 60.9 kDa protein that is required for maturation of the 35S primary transcript of pre-rRNA and is required for cleavages leading to mature 18S RNA. RRP3 was identified in a PCR screen for DEAD box genes. DEAD box genes are part of a large family of proteins homologous to the eukaryotic transcription factor elF-4a. Most of these proteins are RNA-dependent ATPases and some of them have RNA helicase activity. This is the third yeast DEAD box protein that has been shown to be involved in rRNA assembly, but the only one required for the processing of 18S RNA. Mutants of the two other putative helicases, Spb4 and Drsl, both show processing defects in 25S rRNA maturation. In strains where Rrp3 is depleted, 35S precursor RNA is improperly processed. Cleavage normally occurs at sites A0O, Al and A2, but in the Rrp3 depletion stain cleavage occurs between A2 and B1. Rrp3 has been purified to homogeneity and has a weak RNA-dependent ATPase activity which is not specific for rRNA.

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.