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The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

The RecA protein of Escherichia coli is important for genetic recombination in vivo and can promote synapsis and strand exchange in vitro. The DNA pairing and strand exchange reactions have been well characterized in reactions with circular single strands and linear duplexes, but little is known about these two processes using substrates more characteristic of those likely to exist in the cell. Single-stranded linear DNAs were prepared by separating strands of duplex molecules or by cleaving single-stranded circles at a unique restriction site created by annealing a short defined oligonucleotide to the circle. Analysis by gel electrophoresis and electron microscopy revealed that, in the presence of RecA and single-stranded binding proteins, a free 3' homologous end is essential for stable joint molecule formation between linear single-stranded and circular duplex DNA. (2) have shown that the formation of joint molecules occurs in such a fashion that only the 5' end of the viral (+)-strand is displaced from the duplex DNA (Fig. 1B). Using the same substrates, Cox and Lehman (3) confirmed this polarity by restriction endonuclease analysis of RecApromoted branch migration. West et al. (4) concluded that the polarity was the same using linearized duplex DNA and homologous circular ss DNA that carried a short hybridized fragment. These data indicate that, in reactions involving linear duplex and ss circular DNA substrates, the polarity of RecA-catalyzed strand exchange in vitro is 5'-to-3' relative to the ss DNA.While the pairing of circular ss DNA with linear duplex DNA is a rapid and efficient reaction whose substrates and products are well characterized, it is not clear what relationship exists between these substrates and those found in vivo. It would be of interest to examine the formation of stablejoint molecules in reactions involving a ss DNA substrate possessing a free end because such a substrate is likely to be more representative of recombinogenic DNA existing in the cell than the ss circular DNAs used in previous in vitro studies (2)(3)(4)(5)(6). In this study, the formation ofjoint molecules between linear ss DNA and circular duplex DNA substrates has been analyzed. These studies reveal that homology at the 3' end of the linear ss DNA is essential for stable RecAcatalyzed joint molecule formation. This observation is consistent with some existing biochemical data (4, 7), which indicate an important role for free homologous 3' ends in recombination. However, it apparently contradicts predictions based on the polarity of strand exchange involving circular ss and linear duplex DNA substrates (2-4): if RecAcatalyzed strand exchange proceeds 5'-to-3' relative to the ss DNA, then linear ss DNAs in which homology to the duplex circle is restricted to the 3' end would form joint molecules that dissociate with time, while those in which homology is present at the 5' end would grow and become more stable.Two possible explanations to resolve this apparent paradox and their relevance to our understanding o...

Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (A_AG $ GUAAGUAdT) is capable of inducing formation of U2/U4/U5/U6 snRNP complex we show that this oligo specifically binds to U4/U5/U6 snRNP and cross-links to U6 snRNA in the absence of U2 snRNP. Moreover, 5'SS RNA oligo bound to U4/U5/U6 snRNP is chased to U2/U4/U5/U6 snRNP complex upon addition of U2 snRNP. Recognition of the 5'SS by U4/U5/U6 snRNP correlates with the 5'SS consensus sequence. Unlike the interaction with U1 snRNP, this recognition depends largely on interactions other than RNA-RNA base pairing. Finally, the region of U6 snRNA required for this interaction with U4/U5/U6 snRNP is positioned upstream of stem I in the U4-U6 structure. We propose that the 5'SS-U4/US/U6 snRNP complex is an intermediate in spliceosome assembly and that recognition of the 5'SS by U4/U5/U6 snRNP occurs after the 5'SS-U1 snRNA base pairing is disrupted but before the U4-U6 snRNA structure is destabilized.[Key Words: 5' splice site recognition; spliceosome assembly~ snRNPs] Received May 13, 1994~ revised version accepted July 1, 1994.Removal of introns from nuclear precursor messenger RNA {pre-mRNA} is mediated by a large multicomponent complex called the spliceosome which is composed of U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs} and numerous proteins (for review, see Green 1991; Guthrie 1991; Moore et al. 1993).In mammals, spliceosome assembly depends on a consensus sequence at the 5' splice site (5'SS}, a branch site with an adjacent polypyrimidine tract, and a 3'SS consensus sequence. Initially, U 1 snRNP binds via base pairing with the 5'SS and commits the pre-mRNA to the splicing pathway. Subsequently, U2 snRNP binds at the branch site to form splicing complex A, which is converted into splicing complex B upon association of U4/ U5/U6 triple snRNP. Within U4/U5/U6 snRNP, U4, and U6 snRNAs are extensively base paired. Prior to {or concomitant with} the first step of splicing, this U4-U6 base pairing is disrupted and U4 snRNP is released. Because the pairing interaction between U6 and U4 snRNAs has been proposed to negatively regulate U6 and is mutually exclusive with the pairing interaction between U6 and U2 snRNAs, this structural rearrangement could represent the catalytic activation of the spliceosome.