A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme.

Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

Ribozyme catalysis from the major groove of group II intron domain 5.

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 canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.

A short 5' splice site RNA oligo can participate in both steps of splicing in mammalian extracts.

A short 5' splice site RNA oligonucleotide (5'SS RNA oligo) undergoes both steps of splicing when a second RNA containing the 3' splice site region (3'SS RNA) is added in trans. This trans-splicing reaction displays the same 5' and 3' splice site sequence requirements as cis-splicing of full-length pre-mRNA. The analysis of RNA-snRNP complexes formed on each of the two splice site RNAs is consistent with the formation of partial complexes, which then associate to form the complete spliceosome. Specifically, U2 snRNP bound to the 3'SS RNA associates with U4/U5/U6 snRNP bound to the 5'SS RNA oligo. Thus, as expected, trans-splicing depends on the integrity of U2, U4, and U6 snRNAs. However, unlike cis-splicing, trans-splicing is enhanced when the 5' end of U1 snRNA is blocked or removed or when the U1 snRNP is depleted. Thus, the early regulatory requirement for U1 snRNP, which is essential in cis-splicing, is bypassed in this trans-splicing system. This simplified trans-splicing reaction offers a unique model system in which to study the mechanistic details of pre-mRNA splicing.

U4/U5/U6 snRNP recognizes the 5' splice site in the absence of U2 snRNP.

Using an in vitro system in which a 5' splice site (5'SS) RNA oligo (AAG decreases 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/U5/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.

Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly.

A short RNA oligonucleotide comprising the 5' splice site consensus sequence (5'SS RNA) is sufficient to bind U1 small nuclear ribonucleoprotein particle (snRNP) or to induce the association of U2 snRNP and U4-U5-U6 triple snRNP. Analysis of the requirements of these interactions demonstrates that the 5'SS RNA is recognized independently by at least two different elements during spliceosome assembly: the 5' end of U1 snRNA and a component(s) of the U2-U4-U5-U6 snRNP complex. Since stable 5'SS RNA-U1 snRNA base pairing prevents interaction of the 5'SS RNA with U2-U4-U5-U6 snRNP complex, we conclude that disruption of the initial base pairing between the 5'SS RNA and the 5' end of U1 snRNA is required for subsequent spliceosome assembly.

ATP hydrolysis and the displaced strand are two factors that determine the polarity of RecA-promoted DNA strand exchange.

When the recA protein (RecA) of Escherichia coli promotes strand exchange between single-stranded DNA (ssDNA) circles and linear double-stranded DNAs (dsDNA) with complementary 5' or 3' ends a polarity is observed. This property of RecA depends on ATP hydrolysis and the ssDNA that is displaced in the reaction since no polarity is observed in the presence of the non-hydrolyzable ATP analog, ATP gamma S, or in the presence of single-strand specific exonucleases. Based on these results a model is presented in which both the 5' and 3' complementary ends of the linear dsDNA initiate pairing with the ssDNA circle but only one end remains stably paired. According to this model, the association/dissociation of RecA in the 5' to 3' direction on the displaced strand determines the polarity of strand exchange by favoring or blocking its reinvasion into the newly formed dsDNA. Reinvasion is favored when the displaced strand is coated with RecA whereas it is blocked when it lacks RecA, remains covered by single-stranded DNA binding protein or is removed by a single-strand specific exonuclease. The requirement for ATP hydrolysis is explained if the binding of RecA to the displaced strand occurs via the dissociation and/or transfer of RecA, two functions that depend on ATP hydrolysis. The energy for strand exchange derives from the higher binding constant of RecA for the newly formed dsDNA as compared with that for ssDNA and not from ATP hydrolysis.

DNA substrate requirements for stable joint molecule formation by the RecA and single-stranded DNA-binding proteins of Escherichia coli.

In reactions between linear single-stranded DNAs (ssDNAs) and circular double-stranded DNAs (dsDNAs), stable joint molecule formation promoted by the recA protein (RecA) requires negative superhelicity, a homologous end, and an RecA-ssDNA complex. Linear ssDNAs with 3'-end homology react more efficiently than linear ssDNAs with 5'-end homology. This 3'-end preference is explained by the finding that 3'-ends are more effectively coated by RecA than 5'-ends, as judged by exonuclease VII protection, and are thus more reactive. The ability of linear ssDNAs with 5'-end homology to react is improved by the presence of low concentrations of exonuclease VII. In reactions between ssDNAs and linear dsDNAs with end homology, stable joint molecule formation occurs more efficiently when the homology is at the 3'-end rather than at the 5'-end of the complementary strand. In addition, linear dsDNAs with homology at the 3'-end of the complementary strand react more efficiently with linear ssDNAs with 3'-end homology than with linear ssDNAs with 5'-end homology. The ability of linear ssDNAs with 5'-end homology to react, in the absence of single-stranded DNA-binding protein, is improved by adding 33-46 nucleotides of heterologous sequence to the 5'-end of the linear ssDNA. The poor reactivity of linear ssDNAs with 5'-end homology is explained by a lack of RecA at the 5'-ends of linear ssDNAs, which is a consequence of the polar association and dissociation of RecA.

The preference for a 3' homologous end is intrinsic to RecA-promoted strand exchange.

The recA protein (RecA) promotes DNA pairing and strand exchange optimally in the presence of single-stranded binding protein (SSB). Under these conditions, 3' homologous ends are essential for stable joint molecule formation between linear single-stranded DNA (ssDNA) and supercoiled DNA (i.e. 3' ends are 50-60 times more reactive than 5' ends). Linear ssDNAs with homology at the 5' end do not participate in pairing. In the absence of SSB, the strand exchange reaction is less efficient; however, linear ssDNAs with 3' end homology are still 5- to 10-fold more reactive than those with 5' end homology. The preference for a 3' homologous end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange. The preferential reactivity of 3' homologous ends is likely to be a consequence of the polarity of polymerization of RecA on ssDNA. Specifically, since RecA polymerizes in the 5'----3' direction, 3' ends are more likely to be coated with RecA and, hence, will be more reactive than 5' ends.

3' homologous free ends are required for stable joint molecule formation by the RecA and single-stranded binding proteins of Escherichia coli.

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.

The ATPase core of a clathrin uncoating protein.

Chymotryptic digestion of bovine brain uncoating ATPase produced a 60-kDa fragment that was subsequently proteolyzed to 44 kDa. Loss of clathrin cage uncoating activity paralleled the conversion of the intact 70-kDa enzyme to the 60-kDa fragment, while clathrin binding activity was lost as the 60-kDa fragment was degraded to 44 kDa. This 44-kDa fragment has been purified to homogeneity and characterized as a clathrin-independent ATPase. The 44-kDa ATPase domain has been localized within the intact enzyme by the use of amino-terminal specific antibodies. This localization relates to the conserved nature of the 70-kDa heat shock protein family, of which bovine brain uncoating ATPase is a constitutively expressed member.