A minor groove RNA triple helix within the catalytic core of a group I intron

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

nature structural biology • volume 5 number 12 • december 1998 1037

Close packing of several double helical and single stranded RNAelements is required for the Tetrahymena group I ribozyme toachieve catalysis. The chemical basis of these packing interac-tions is largely unknown. Using nucleotide analog interferencesuppression (NAIS), we demonstrate that the P1 substrate helixand J8/7 single stranded segment form an extended minorgroove triple helix within the catalytic core of the ribozyme.Because each triple in the complex is mediated by at least one 2'-OH group, this substrate recognition triplex is unique to RNAand is fundamentally different from major groove homo-purine–homopyrimidine triplexes. We have incorporated thesebiochemical data into a structural model of the ribozyme core

that explains how the J8/7 strand organizes several helices withinthis complex RNA tertiary structure.

The Tetrahymena ribozyme folds into a complex tertiary structurewith a densely packed catalytic core (Fig. 1a)1. The ribozyme is com-prised of two components: the P4-P6 domain, whose structure hasbeen solved2, and the P3-P7-P8 element, whose structure is notknown at high resolution. The active site of the intron is formed atthe interface of these two regions, and it is here that the 5'-exon (orP1) helix docks and the chemistry of RNA splicing is catalyzed1. P1docking provides a model system for the study of RNA helix packinginteractions because docking can be monitored by ribozyme activity.

The P1 helix is formed by base pairing of the 5'-exon to a comp-lementary internal guide sequence (IGS) within the intron(Fig. 1a)3. P1 docks into the active site by forming several tertiaryinteractions that involve specific functional groups in the duplex4–7.The highly-conserved G22·U(-1) wobble pair adjacent to the cleav-age site in P1 makes five specific minor groove tertiary interactionswith two consecutively stacked sheared A·A pairs located in the J4/5segment of P4-P68,9.

A minor groove RNA triplehelix within the catalyticcore of a group I intron

Fig. 1 a, Secondary structure and modeled regions of the Tetrahymenaintron. The sequence and location of nucleotides discussed in the text areshown with their nucleotide numbers. The crystal structure of the P4-P6domain (blue) has been determined2. P1 (gray), J8/7 (green) and P3(magenta) are the secondary structural elements that are modeled in thiswork. Red boxes mark the residues in P1 that interact with J8/7 throughtheir 2'-OH groups. Heavy lines show the schematic secondary structure ofthe intron, and thin lines illustrate the connectivity between sequenceelements. The P2-2.1 and P9 extensions have been omitted for clarity. b, Identification of tertiary interactions by interference suppression. TheL-21 G414 form of the ribozyme performs a 3'-exon ligation reaction thattransfers the 3'-end of an oligonucleotide substrate (A*) onto the 3'-ter-minal G of the ribozyme. For simplicity, a ribozyme is depicted with justfive A’s, three of which participate in critical tertiary hydrogen bonds. In aNAIM experiment an α-phosphorothioate tagged 2'-deoxyadenosineanalog is randomly, but individually, incorporated at each of the five A’s inthe context of a wild type RNA sequence. The presence (red) or lack(green) of interference is determined by the disappearance of the corre-sponding band in the RNA sequencing gel (right). In this example, dAαSinterference suggests that the 2'-OHs at positions 1, 3 and 4 are essentialfor 3'-exon ligation activity, while positions 2 and 5 are not. An NAISexperiment is performed in the same manner as NAIM, but G25 has beenuniformly replaced with 2'-deoxyguanosine (blue). This eliminates one ofthe critical hydrogen bonds (blue circle) and causes all of the molecules inthe population to be less active. In this example, dAαS interference map-ping in the context of dG25 substitution results in specific interferencesuppression at A4 (see gel at right). This implicates the 2'-OH of A4 as thehydrogen bonding partner for G25. Interference at A1 and A3 are notsuppressed, even though position A3 also makes tertiary contact with theP1 helix. This approach has been used to define potential tertiary interac-tion partners throughout the group I catalytic core.

a

b


199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

1038 nature structural biology • volume 5 number 12 • december 1998

b

d

a

Fig. 2 NAIS experiments to define tertiary interaction partners for 2'-OH groups in the IGS. a, Interference mapping within the J8/7 regionof the intron. The 3'-exon ligation and 5'-end labeled control lanes areshown for ribozymes with single site substitutions in the IGS (ribozymesubstitution indicated above the bracket) and an adenosine analograndomly incorporated throughout the RNA (analog identity shownabove each lane). The nucleotide numbers that correspond to thebands in the sequence are shown to the left and right. Additional elec-trophoretic runs using the same experimental samples were performedto resolve other informative regions of the sequence, such as A207 andA114. A302 and A306 are not detected in this 3'-ligation reactionbecause of a strong phosphorothioate effect at these two sites. The ori-entation of the 5'-end labeled control has been inverted for ease ofcomparison. b–d, Graphs of nucleotide analog interference at sitesthroughout the intron for each analog (indicated at left) and eachribozyme (indicated at top). The bar height indicates the magnitude ofthe interference9. A value of 1 indicates no interference, a value >2indicates moderate interference, and a value of 6 indicates stronginterference at that site. Asterisks highlight the combination of phos-phorothioate analog and IGS substitution that result in interferencesuppression. The values represent the average of two independentexperiments with an error of ±20%.

c


199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters


Four other sequence non-specific 2'-OH groups located on the5'-exon and IGS strands also participate in P1 helix docking4–6. Thecritical 2'-OH groups include those at C(-2) and U(-3) on the 5'-exon strand and G25 and G26 on the IGS strand (Fig. 1a). In anA-form RNA duplex all four of these 2'-OHs lie on the same face ofthe helical minor groove, about a quarter turn away from the exo-cyclic amine of the G22·U(-1) pair. The tertiary hydrogen bondingpartner for only one of these 2'-OH groups has been identifiedwithin the P3-P7-P8 domain10. Mutagenesis and dimethylsulfate(DMS) footprinting experiments demonstrated that the U(-3) 2'-OH donates a hydrogen bond to the N1 of A302, which is locat-ed in the J8/7 single stranded region of the core. Identification ofthe tertiary hydrogen bonding partners for all four 2'-OH groups inP1 would provide valuable insight into the complex tertiary struc-ture at the core of this catalytic RNA.

J8/7 is a particularly critical segment of the group I active site. Itis single stranded due to the formation of the P3 pseudoknot, andthe seven nucleotides that comprise its length include several of themost phylogenetically conserved positions within the intron11. Inaddition to binding the P1 duplex10, J8/7 interacts with the P4helix12, contacts the P3 helix9,13, is in proximity to the G binding sitewithin the P7 helix14, and is covalently connected to the P8 and P7helices15. Based upon nucleotide analog interference suppression(NAIS) experiments, we demonstrate that the specific tertiaryinteraction partners for all four 2'-OH groups in P1 distal from thecleavage site are within the J8/7 segment. These restraints are usedto generate a model of the group I intron catalytic core thatincludes a minor groove triple helix between J8/7 and P1.

The 2'-OHs of G25 and A301 interactThe G25 2'-OH contributes significantly to P1 helix docking (>2 kcal mol–1)6,16. It was initially proposed to hydrogen bond withthe N1 of A30117, but such an interaction proved inconsistent withdimethyl sulfate footprinting results (S.A.S. and T.R. Cech, unpub-lished data). Nucleotide analog interference mapping (NAIM), a

chemogenetic method used to efficiently identify the chemicalgroups responsible for RNA activity (Fig. 1b)18,19, demonstratedthat two other functional groups of A301, the 2'-OH and N6, areimportant for intron function and that there is close helix packingagainst the minor groove edge of J8/7 at A3019.

To determine if the hydrogen bonding partner for the G25 2'-OH is the 2'-OH of A301, we performed NAIS, an extension ofNAIM, which makes it possible to identify the interacting function-al groups within an RNA tertiary structure20 (Fig. 1b). We preparedL-21 G414 Tetrahymena group I ribozymes with a single 2'-deoxyguanosine substitution at position 25 (dG25 ribozyme)and either AαS, dAαS, FAαS, or DAPαS substitutions randomlyincorporated throughout the length of the RNA (see Table 1 forabbreviations)6,21. In the context of the wild type ribozyme, dAαS,FAαS, and DAPαS each show strong interference at A301 and sev-eral other positions in the intron9. The dG25 substitution resultedin selective interference suppression at A301 for the analogs dAαSand FAαS, both of which modify the 2'-OH group (Fig. 2a, com-pare lanes 3 and 4 with lanes 6 and 8; Fig. 2b). dG25 substitutiondid not suppress DAPαS (an analog that adds an extra N2 aminogroup to A) interference anywhere within the molecule, includingA301 (Fig. 2a, lane 2; Fig. 2b). Thus, dG25 interference suppressionat A301 is specific for the position within the sequence and thefunctional group alterations introduced by the nucleotide analog.

As additional controls for the specificity of dG25 interferencesuppression, we prepared ribozymes containing either a single dGor inosine substitution (I; a G analog that deletes the N2 amine) atthe G22 wobble position (dG22 or I22 ribozymes) and randomlyincorporated AαS or dAαS throughout the length of the RNA.These two G22 substitutions delete functional groups that hydro-gen bond with the J4/5 wobble receptor, and result in interferencesuppression within that region of the catalytic core8. As expected,neither the G22 nor the I22 ribozymes suppressed dAαS interfer-ence at A301 (Fig. 2a, lanes 6 and 8; Fig. 2b). This indicates thatsuppression of dAαS interference is specific to the dG25 ribozyme

Fig. 3 Model of the elements surrounding the J8/7 region of the Tetrahymena group I intron catalytic core. a, A minor groove triple helix betweenthe J8/7 single stranded segment (green) and the P1 helix (gray). The four hydrogen bonds defined by biochemical experiments that anchor P1against J8/7 are indicated with dashed red lines. A possible fifth hydrogen bond implied by the data is shown in blue. The scissile phosphate at the3'-position of U(-1) is brown. b, A model of the portion of the group I intron catalytic core that contains close packing interactions between J8/7 andhelices P1, P3 and P4. It also includes the wobble–wobble receptor interaction between P1 and J4/5. The hydrogen bonding interactions used as mod-eling constraints are shown as dashed red lines. A complete list of these interactions is given in Table 2. The color scheme for the individual RNA seg-ments is the same as in Fig. 1a. The scissile phosphate is brown except for the 3'-oxygen leaving group, which is shown as a red sphere. The fivefunctional groups that may act as leaving group metal ion ligands are shown as yellow spheres.

a b


199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters


and rules out interference suppression due to global undocking ofthe P1 helix.

These data indicate that the G25 2'-OH is energetically coupled tothe 2'-OH of A301. The nature of the interaction is most likely by adirect hydrogen bond between these groups, although the possibili-ty of a water-mediated interaction cannot be completely discount-ed. The fact that FAαS substitution causes interference at A301suggests that its 2'-OH donates a hydrogen bond to the 2'-oxygen of G259. This would allow the G25 2'-OH to donate a sec-ond hydrogen bond to the N3 of A301, forming a ribose zipper. Allthree of the expected hydrogen bonds can be readily accommodatedby docking an A-form A301-A302 dinucleotide into an A-form P1helix (Fig. 3a). Because G25 and U(-3) are located on opposingstrands of the P1 helix, the experimental data indicate that these A’sbridge across the P1 minor groove at an angle of ~40° (Fig. 3a).

Interaction partners for the G26 and C(-2) 2'-OHs in J8/7The C(-2) and G26 2'-OH groups also contribute to P1 docking,although each donates less than 1 kcal mol–1 of free energy5,6,16.These two nucleotides flank each side of the interaction surfacemade between the A301-A302 dinucleotide and the P1 helix.Extension of the dinucleotide model by one residue in both the 5'and 3' directions using A-form helical geometry places nucleotidesU300 and G303 into a reasonable position for these nucleotides tomake additional direct hydrogen bonds to P1. In such a model, the2'-OH of G26 appears to be within hydrogen bonding distance ofthe 2'-OH of U300, and the 2'-OH of C(-2) could pair with eitherthe N2 exocyclic amine or the 2'-OH of G303. All three of thesefunctional groups in J8/7 are important for intron activity asdefined by NAIM (Fig. 2c, 2d). There is strong dUαS interference atU300, which demonstrates the importance of the 2'-OH (Fig. 2c),and dGαS, IαS, and m2GαS (Table 1) all show strong interference atG303, which implies a role for the 2'-OH and N2 amine(Fig. 2d)19,22.

The possibility of an interaction between the 2'-OHs of G26 andU300 was tested using four dG26 ribozymes with either UαS,dUαS, AαS, or dAαS incorporated throughout the length of theRNA. As an additional control we also prepared two dG25ribozymes with UαS or dUαS analogs incorporated. As predictedby the simple A-form model, dUαS interference at U300 wasspecifically suppressed by the dG26 substitution, and it was notsuppressed by the dG25 substitution (Fig. 2c). Furthermore, dG26substitution did not affect any of the sites of dAαS interference,including that at A301. These data argue for a tertiary interactionbetween the 2'-OH of U300 and the 2'-OH of G26. Furthermore,U300 interference from dUαS, but lack of interference from FUαS(data not shown) suggests that the U300 2'-OH acts as a hydrogenbond acceptor from the 2'-OH of G26 (Table 2).

The final 2'-OH that is important for P1 docking is at C(-2),which appears to be in position to hydrogen bond with either the

2'-OH or N2 amine of G303. To test for such an interaction, NAISexperiments were performed using a substrate with a C(-2) 2'-deoxy modification (substrate dC(-2)dT(-1)S) and ribozymescontaining random GαS, dGαS, IαS, m2GαS, AαS or dAαS substi-tutions. We found that the dC(-2) substitution specifically sup-pressed IαS interference at G303 (Fig. 2d), moderately suppressedG303 interference from m2GαS, but did not suppress dGαS inter-ference. It did not suppress any of the sites of dAαS interference,including A301. This suggests that the N2 exocyclic amine and notthe 2'-OH of G303 forms a hydrogen bond to the 2'-OH of C(-2).

An RNA minor groove triple helixWe included the G26–U300 and C(-2)–G303 tertiary contacts asadditional geometric constraints to generate the P1-J8/7 dockingmodel shown in Fig. 3a (see also Table 2) In this model, the J8/7region forms an extended four base pair triple helical complex inthe minor groove of P1. All five of the hydrogen bonds can beaccommodated using an A-form geometry for both P1 and J8/7.Every hydrogen bond is mediated by at least one 2'-OH group, sothis triplex could only be formed by an RNA molecule. The J8/7strand interacts with both strands of the P1 helix. It binds twonucleotides at the 3' end of one strand, crosses the minor grooveand binds two nucleotides at the 3'-end of the other strand. Thisdyad symmetry means that there is no requirement for the absoluteorientation of the helix relative to the third strand. All the criticalfunctional groups within the P1 helix are sequence non-specific 2'-OH groups, but three of the five hydrogen bonding groups in thethird strand are particular to the J8/7 sequence and define a 5'-NAAG-3' consensus sequence (Table 2).

This RNA minor groove triplex is completely different from thetriple helices reported for homopurine–homopyrimidinesequences in DNA23,24. In those structures, the third strand isbound in the major groove and oriented parallel to the homop-urine strand that it binds23,24. The only common structural featurebetween these two classes of triplexes is that they both involve threestrands. It is also noteworthy that the tertiary interactions identi-fied in this study were not predicted by the group I intron modelsgenerated by Michel, Westhof and coworkers13,25.

A model of the group I active siteIn addition to binding the P1 helix, J8/7 has been shown to makeinteractions with several other segments of the catalytic core. It iscovalently connected to helices P7 and P8. Residue U305 makes amajor groove base triple with the P4 helix via the G111-C209 basepair12. This interdomain tertiary interaction is readily accommo-dated into the model by extending J8/7 to include nucleotides A304and U305 (Fig. 3a,b). Within the model, J8/7 is in position to makethe tertiary interaction with the major groove of P4, thoughresidues A304 and U305 must be distorted from A-form geometry

Table 1 Nucleotide analogs

(Nucleotide) analog Abbreviation, phosphorothioate tagged form Functional group change(s)(A) 2'-Deoxyadenosine dAαS 2'-OH replaced with H(A) Diaminopurine riboside DAPαS Amino group added at C2 position(A) Purine riboside PurαS N6 exocyclic amine replaced with H(A) 2'-Deoxy-2'-fluoro adenosine FAαS 2'-OH replaced with fluorine(G) 2'-Deoxyguanosine dGαS 2'-OH replaced with H(G) N2-Methylguanosine m2GαS N2 exocyclic amine methylated(G) Inosine IαS N2 exocyclic amine replaced with H(U) 2'-Deoxyuracil dUαS 2'-OH replaced with H(U) 2'-Deoxy-2'-fluoro uracil FUαS 2'-OH replaced with fluorine


199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters


(Fig. 3b)9. J8/7 also makes a direct tertiary contact with the P3 helixwhere it forms a U300·A97–U277 base triple9,13 (M. Zivarts and S.A.S., unpublished results). Modeling of this interaction indicates thatthere is close packing of P3 against both J8/7 and the minor groove ofP1 (Fig. 3b). This suggests that U300 and possibly A301 make directhydrogen bonding contacts to two helices, and that this portion ofthe J8/7 single stranded segment serves as a spine upon which bothP1 and P3 are aligned for close packing in the catalytic core.

All of these interactions can be readily incorporated into a singlemodel of the group I intron active site, which results in a complexmulti-layered structure that is stabilized by an interlocking networkof hydrogen bonds (Fig. 3b). The model provides a completedescription of all the ground state interactions between P1 and thecatalytic core, including tertiary interactions with two distinctregions of the intron, J4/5 and J8/7. In addition to the interactionwith P1, J8/7 makes isolated base triples with P3 and P4, and spansbetween P8 and P7. In so doing, this single stranded region facili-tates the close approach of no fewer than five different helical ele-ments within the core of the group I intron.

Potential structural and catalytic metal binding sitesThe group I intron is a metalloenzyme that requires divalent metalions for folding and catalysis26, so the issue of phosphate charge anddivalent metal binding must be considered. Phosphorothioateinterference has been observed at several residues included withinthe triple helix model9,27,28, which is indicative of phosphate–RNAor phosphate–metal ion interactions. Several of the strongest phos-phorothioate effects within the intron are at J8/7 positionsA302–A306, inclusive9,28. When all of the P4-P6 structure is includ-ed in the model, the pro-RP oxygens of A302 and G303 are close tothe J6/7 phosphate oxygens of U258 and U259, which also demon-strate strong phosphorothioate effects28. Interference at all fourpositions can be partially rescued by addition of Mn2+, a ‘soft’ Lewisacid that can bind to the non-bridging sulfur9,28. One interpreta-tion of these data is that a divalent metal ion may bridge betweenthe phosphate backbones of J8/7 and J6/7.

The other three sites of phosphorothioate interference in J8/7(A304, U305 and A306) are of particular interest because of theirproximity to the scissile phosphate. Biochemical experiments havedemonstrated that at least two divalent metals stabilize the transes-terification reaction catalyzed by the group I intron29,30. One metal

activates the 3'-nucleophile of G, and a second metal stabilizes the3'-oxyanion leaving group of U(-1). In the P1 docking model thereare five functional group candidates that may participate in themetal binding site near the 3'-oxygen of U(-1) (yellow balls, Fig.3b). These include the pro-RP oxygens of C208, A304 and U305, thepro-SP oxygen of A207, and the 2'-OH of G303. All of these groupshave been implicated as important for group I activity9,28,31,although additional experiments will be needed to test this propos-al. Because this model does not include the P7 helix and the Gbinding site, we cannot comment on the location of the othermetal.

Our model suggests a framework for 5'-splice site selection in thegroup I intron. The P1 helix is docked into the catalytic core againsttwo structural elements, J4/5 and J8/7. The G·U wobble interactionwith J4/5 is the only sequence specific contact that is made betweenP1 and the catalytic core and it largely defines the 5'-splice site8. Bycontrast, J8/7 only makes non-sequence specific interactions withP1. These interactions stabilize binding of the substrate helix andare likely to align the catalytic metal ions against the phosphateselected for cleavage by J4/5. In this way, two separate functionaldomains act in concert to effect accurate and efficient splicing.

MethodsRibozyme preparation. NAIS experiments to explore the tertiaryinteractions with the G25 and G26 2'-OHs were performed using a totalof 14 full-length L-21 G414 semisynthetic RNAs prepared by ligating a17-mer oligonucleotide onto an L-38 G414 RNA as described for relatedRNAs6. AαS, dAαS, FAαS, or DAPαS were incorporated into the L-38G414 transcripts as described9. dUαS was incorporated using 0.5 mMdUTPαS in the presence of 1 mM NTPs by the Y639F T7 RNA polymerasepoint mutant. NAIS experiments to explore interactions with the 2'-OHat C(-2) were performed using an oligonucleotide substrate with the 2'-deoxy C(-2) substitution, dC(-2)dT(-1)S [5'-CCCU(dCdT)AAAAA-3’],and fully transcribed L-21 G414 RNAs containing random IαS, m2GαS,dGαS, PurαS, dAαS, or DAPαS substitutions9,19,22.

Interference suppression. Oligonucleotide substrates rT(-1)S [5'-CCCUC(rT)AAAAA-3'], dT(-1)S [5'-CCCUC(rT)AAAAA-3'], or dC(-2)dT(-1)Swere radiolabeled at their 3'-ends with [α-32P]cordycepin ([α32P]3'-deoxyATP). Interference suppression resulting from deletion of theG25 2'-OH was assayed by reacting the enzymes shown in Fig. 2a withthe substrate rT(-1)S for 1 min. at 30 °C. G26 interference suppres-sion was assayed using ribozymes listed in Fig. 2b with the oligonu-cleotide dT(-1)S for 30 min at 30 °C. Effects of deleting the 2'-OH atC-2 were determined using ribozymes in Fig. 2c with the substratedC(-2)dT(-1)S at 30 °C for 10 min. Reaction conditions were selectedthat gave a significant percentage (~20%) of intron-exon ligationproduct, but which still showed interference at most sites through-out the intron. L-21 G414 RNAs were radiolabeled at their 5'-ends tocontrol for the extent of analog incorporation at each position9.Peak intensities for both the parental nucleotide (AαS, GαS, or UαS)and the nucleotide analog were quantitated by PhosphorImageranalysis at each position in the 3'-ligation experiment and the 5'-endlabeled control9 and used to calculate the magnitude of interference.Due to errors inherent in quantitation of band intensities, any inter-ference effects that were quantitated as being greater than six-foldwere defined as having a magnitude of 6.

Modeling of the Tetrahymena intron core. The P4-P6 domain crys-tal structure with added hydrogens was used as the starting point formodeling additional elements within the Tetrahymena group I introncore. A P1 helix model with the Tetrahymena sequence from C(-6) toA(+1) and from G22 to G27 was built with A-form geometry and G·Ucoordinates from the G148·U155 base pair found within the P4-P6domain2, and docked into J4/5 following the original G·U receptormodel8. Simulated annealing molecular dynamics calculations wereemployed to create an energy-minimized model for the P1, P3, J8/7and P4-P6 elements. After minimizing the U300–A304 region of themodel with hydrogen bonding restraints between J8/7 and P1(Table2), P3 was placed in proximity to U300 and a second round of simulat-

Table 2 Pairwise tabulation of interacting functional groups in P4,P1, J8/7 and P3 used to model the Tetrahymena intron active site

Residue Functional group Residue Functional groupJ4/5 Loop P1 HelixA207 N3 G22 HN2A207 O2' G22 HN2A114 N3 G22 HO2'A114 HO2' G22 O2'C208 HO2' G23 O2'J8/7 Strand P1 HelixU300 O2' G26 HO2'A301 N3 G25 HO2'A301 HO2' G25 O2'A302 N1 U(-3) HO2'G303 HN2 C(-2) O2'J8/7 Strand P3 HelixU300 H3 A97 N7U300 O4 A97 HN6J8/7 Strand P4 HelixU305 O4 C209 HN4


199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters


Structure of a human DNArepair protein UBA domainthat interacts with HIV-1 Vpr

ed annealing was done with additional restraints that define the U305and U300 base triples (Table 2). During all calculations the P4-P6domain and the docked P1 helix were held rigid. In the resultingmodel, there are no van der Waals or chemical geometry violations.

Note added in proof: While this work was in press, Golden et al.32

published a model based upon 5 Å crystallographic data of aTetrahymena intron lacking the P1 helix and several other periph-eral elements known to stabilize the catalytic core. Within the J8/7region there are substantial discrepancies between the 5 Å modeland the results presented here as well as other biochemical data9,10.In order for J8/7 to form the appropriate base triples with P1 andP3, it must rotate 180° about the phosphate backbone at A301 andA302 and P3 must translate by more than 20 Å near U300. Thissuggests that the J8/7 region of the crystallized intron lacking the P1helix may not be preordered for substrate binding.

AcknowledgmentsWe thank R. Sousa for the clone of the Y639F mutant T7 RNA polymerase, R.R. Gutellfor analysis of group I sequences, and L. Weinstein for helpful comments on themanuscript. S.P.R. was supported by an NIH training grant. This work was funded byan NIH grant, a Beckman Young Investigator Award, a Searle Scholar award, and aJunior Faculty Research Award from the American Cancer Society to S.A.S.

Alexander A. Szewczak, Lori Ortoleva-Donnelly, Sean P.Ryder, Eileen Moncoeur and Scott A. Strobel

Department of Molecular Biophysics and Biochemistry, 260 Whitney Avenue, YaleUniversity, New Haven, Connecticut 06520, USA.

Correspondence should be addressed to S.A.S. email: [email protected]

Received 22 June, 1998; accepted 14 September, 1998.

1. Cech, T.R. & Herschlag, D. In Catalytic RNA, vol. 10 (eds Eckstein, F. & Lilley, D.M.J.)1–17 (Springer, New York; 1996).

2. Cate, J.H. et al. Science 273, 1678–1685 (1996).3. Waring, R.B., Towner, P., Minter, S.J. & Davies, R.W. Nature 321, 133–139 (1986).4. Bevilacqua, P.C. & Turner, D.H. Biochemistry 30, 10632–10640 (1991).5. Pyle, A.M. & Cech, T.R. Nature 350, 628–631 (1991).6. Strobel, S.A. & Cech, T.R. Biochemistry 32, 13593–13604 (1993).7. Strobel, S.A. & Cech, T.R. Science 267, 675–679 (1995).8. Strobel, S.A., Ortoleva-Donnelly, L., Ryder, S.P., Cate, J.H. & Moncoeur, E. Nature

Struct. Biol. 5, 60–66 (1998).9. Ortoleva-Donnelly, L., Szewczak, A.A., Gutell, R.R. & Strobel, S.A. RNA 4, 498–519 (1998).

10. Pyle, A.M., Murphy, F.L. & Cech, T.R. Nature 358, 123–128 (1992).11. Damberger, S.H. & Gutell, R.R. Nucleic Acids Res. 22, 3508–3510 (1994).12. Tanner, M.A., Anderson, E.M., Gutell, R.R. & Cech, T.R. RNA 3, 1037–1051 (1997).13. Michel, F. & Westhof, E. J. Mol. Biol. 216, 585–610 (1990).14. Wang, J.F. & Cech, T.R. Science 256, 526–529 (1992).15. Waring, R.B., Davies, R.W., Brown, T.A. & Scazzocchio, C. Gene 28, 277–291 (1984).16. Narlikar, G.J., Khosla, M., Usman, N. & Herschlag, D. Biochemistry 36, 2465–2477

(1997).17. Michel, F. & Westhof, E. Nature Struct. Biol. 1, 5–7 (1994).18. Conrad, F., Hanne, A., Gaur, R.K. & Krupp, G. Nucleic Acids Res. 23, 1845–1853 (1995).19. Strobel, S.A. & Shetty, K. Proc. Natl. Acad. Sci. USA 94, 2903–2908 (1997).20. Strobel, S.A., Ortoleva-Donnelly, L., Ryder, S.P., Cate, J.H. & Moncoeur, E. Nature

Struct. Biol. 5, 60–66 (1998).21. Moore, M.J. & Sharp, P.A. Science 256, 992–997 (1992).22. Ortoleva-Donnelly, L., Kronman, M. & Strobel, S.A. Biochemistry 37, 12933-12942

(1998).23. Moser, H.E. & Dervan, P.B. Science 238, 645–650 (1987).24. Beal, P.A. & Dervan, P.B. Science 251, 1360–1363 (1991).25. Lehnert, V., Jaeger, L., Michel, F. & Westhof, E. Chem. Biol. 3, 993–1009 (1996).26. Pyle, A.M. Science 261, 709–714 (1993).27. Christian, E.L. & Yarus, M. J. Mol. Biol. 228, 743–758 (1992).28. Christian, E.L. & Yarus, M. Biochemistry 32, 4475–4480 (1993).29. Piccirilli, J.A., Vyle, J.S., Caruthers, M.H. & Cech, T.R. Nature 361, 85–88 (1993).30. Weinstein, L.B., Jones, B.C.N.M., Cosstick, R. & Cech, T.R. Nature 388, 805–808 (1997).31. Beren, C., Steicher, B., Schroeder, R. & Hillen, W. Chem. Biol. 5, 163–175 (1998).32. Golden, B. L.,Gooding, A. R., Podell, A. R. & Cech, T. R. Science 282, 259–264 (1998).

The HIV-1 protein Vpr is critical for a number of viral func-tions including a unique ability to arrest T-cells at a G2/Mcheckpoint and induce subsequent apoptosis. It has beenshown to interact specifically with the second UBA (ubiqui-tin associated) domain found in the DNA repair proteinHHR23A, a highly evolutionarily conserved protein. Thisdomain is a commonly occurring sequence motif in somemembers of the ubiquitination pathway, UV excision repairproteins, and certain protein kinases. The three dimension-al structure of the UBA domain, determined by NMR spec-troscopy, is presented. The protein domain forms acompact three-helix bundle. One side of the protein has ahydrophobic surface that is the most likely Vpr target site.

Vpr mediates a number of different functions during theHIV life cycle1, including nuclear import2 and the inductionof cell cycle arrest in the G2 phase3–6. While the exact mecha-nism of Vpr in induction of cell cycle arrest has not yet beenelucidated, it has been shown that Vpr is both necessary andsufficient to cause accumulation of cells in G2 phase3. Thearrested HIV-1 infected T-cells subsequently undergo celldeath by apoptosis. It has been hypothesized that this canhave a significant effect in HIV pathogenesis by preventingefficient activation and subsequent clonal expansion of CD4+

T-cells7,8. The Vpr mediated cell cycle arrest is in some aspectssimilar to the arrest of cells at certain cell cycle checkpoints asa result of exposure to DNA damaging agents9. Using a yeasttwo-hybrid assay, it has recently been shown that Vpr alsointeracts with the DNA repair protein HHR23A10, the humanhomolog of Rad23. HHR23A is an acidic 40,000 Mr proteinthat shares extensive homology with the protein encoded bythe Saccharomyces cerevisae RAD23 gene11. The Rad23 proteinis involved in nucleotide excision repair and is up-regulated ifthe cell is exposed to DNA-damaging agents11. All of theRad23 homologs identified to date share a common domainstructure with a so-called ubiquitin-like region at the N-ter-minus and two highly conserved regions of ~50 amino acids,termed the ubiquitin associated (UBA) domains12, in the cen-ter and at the C-terminus (Fig. 1a). The 45 amino acid C-ter-minal UBA domain (UBA(2)) was identified as theVpr-interacting domain of HHR23A by an in vitro bindingassay10. UBA domains are a very common sequence motif inproteins of the ubiquitination pathway, UV excision repairproteins and certain protein kinases12. Although the specificrole of this protein domain is so far not known, it has beensuggested that UBA domains are involved in conferring targetspecificity to multiple enzymes of the ubiquitination system.In order to further our understanding of the interaction of theUBA domain with Vpr and other proteins, we have deter-mined the structure of UBA(2) of HHR23A using homonu-clear NMR spectroscopy. This is the first three-dimensionalstructure of a UBA domain.

Withers-Ward et al.10 have shown that overexpression ofboth the full-length HHR23A protein and a 179 amino acid

Documents

A minor groove RNA triple helix within the catalytic core of a group I intron