J. Mol. Biol. (1990) 213, 199-201

COMMUNICATIONS An RNA Holliday Junction? Structural and Dynamic Considerations of the Bacteriophage T4 Gene 60 Interruption

Donald H. Burke-Agiiero and John E. Hearst

Department of Chemistry University of California, Laboratory of Chemical Biodynamics Lawrence Berkeley Laboratory, Berkeley, CA 94720, U.S.A.

(Received 20 June 1988; accepted 20 November 1989)

A novel interrupted gene motif has been reported in which a 50-nucleotide insertion into bacteriophage T4 gene 60 appears to be present in the message at the time of translation, yet it is not translated. We present here a dynamic model for how translation may be occurring in the neighborhood of the interruption. The model involves formation of an RNA structure with similarities to a Holliday junction, followed by migration of the branch point in a strand exchange between message and interruption. The advantage of this model over previous ones is that at no time is a new tRNA required to pair with a discontinuous template.

Huang et al. (1988) have reported a novel gene tion; the order of events is not important to this interruption motif in gene 60 of bacteriophage T4, model. More extensive branch migration is hindered in which a 50-nucleotide intervening segment is by the lack of sequence homology between the neither removed nor translated. The sequence of the message and the dumbbell outside these five bases. interruption suggests that it can be folded to bring When tRNA G~y moves into the P site, the template the translated codons on either side of the inter- 3' to Gly46 will now be in position to receive the ruption into close proximity (Fig. 1). The authors next tRNA (Fig. 2(c)) without having to do so in suggest that bridging the gap may enable the ribo- the presence of the discontinuity of a Holliday-like some to skip across it. On the basis of both the branch point. structural and dynamic potentials of this RNA The structure shown in Figure 1 is related to a sequence, we are suggesting an alternative dynamic family of structures known as gapped dumbbells, in model for readthrough across the interruption which two hairpins stack on one another. Not all involving branch migration of a Holliday-like junc- gapped nucleic acid dumbbells are equally stable. tion between tRNA and mRNA. Nuclear magnetic resonance studies of a DNA One of the most striking features of the gene 60 dumbbell similar to Figure 3(a) show that the interruption is the direct repeat of the sequence exchange rate with solvent of the imino protons 5' UGGAU 3' at each end of the RNA dumbbell. immediately adjacent to the gap is the same as the This duplication is required in order for translation rate for bases more internal to the stem of either to continue beyond the interruption (Weiss et al. hairpin (L. Rinkel & I. Tinoco Jr, personal com- unpublished results). The duplication places the two munication), implying: (1) that there is no fraying of strands containing the pentanucleotides in position the helix at the gap; and (2) that the two helices are to exchange strands with each other, as in the stacked onto one another as though they were one Holliday-like junction shown in Figure 2(a). long helix. Presumably, the same structure can form Branch migration of this junction would permit with equivalent stability in RNA. each succeeding tRNA in. the vicinity of Gly46 to The structure shown in Figure 3(b), in which an pair with a continuous template without the discon- extra base-pair is added across the gap, cannot be tinuity of the gap in Figure 1. While the tRNA made in molecular models without unpairing of the encoding Gly46 is paired with the message stacked helix due to bond length constraints. (Fig. 2(a)), the branch point in front of them can However, if the protruding bases are not required to migrate to a position internal to the pentanueleotide pair, as in Figure 3(c), their presence may or may repeat (Fig. 2(b)). This event may take place either not lead to fraying at the center of the dumbbell. before or after aminoacyl transfer and transloea- The protruding bases may even, in some cases,

0022-2836/90/1 O0199--03 $03.00/0 199 © 1990 Academic Press Limited 200 D. H. Burke-Agiiero and J. E. Hearst

5' U \ ° C (o) (b) G C,,,,,,,,,,,,O Gp4s~U C~G As G~C (c) (d) ~G A~U Figure 3. Relative stabilities of gapped nucleic acid dumbbells. (a) A simple gapped dumbbell. All bases are Gly 4s ~.A,-.U~ A stacked as though it were not gapped. (b) This structure cannot form, because new base-pairs will not form across U the gap without disrupting the stack. (c) If the protruding Leu4 A~ G A bases are not required to pair, it is not known whether 0 UUU. G ~C A their presence will cause fraying of the stack. (d) The ~-A G" U A -,~ A protruding arms are stabilized by base-pairing with A,'U,,~A U another strand. In this case, the bridging strand is Gly 4s/G G U ~" discontinuous. C U = A"C C U A=U U fraying of the 5' stem-loop exposes the stop codon u=A just inside the folded region, which may lead to / premature termination of translation; and (2) the U=A Gly46 codon in the translated portion of the 3' message before the interruption would no longer be A=U physically next to the Leu47 codon in the second G--U half of the message. The pairing of the anticodon loops on either side A A of the gap (Fig. 3(d)) could potentially act to tie G A together the free ends protruding from the dumb- Figure 1, Amino acid sequence and possible message bell, forming a Holtiday-like junction. Like the well- secondary structure of the T4 gene 60 message from characterized DNA junctions, this one would consist nucleotide 514 to nucleotide 575, as presented by Huang of two helices linked by the crossing over of one et al. (1988) as modified by Larry Gold to take note of the strand from each side, forming a branch point GAAA "organizer" motif (personal communication). capable of moving up or down the two helices. Figure 3(d) is unlike the normal Holliday junction, however, in that the fourth (tRNA) strand is not invade the duplex to form a triple-stranded struc- covalently continuous, allowing for greater confor- ture (Michel etal., 1989). This question awaits mational freedom. Although it is believed from further study. Disruption of the analogous helices in nuclear magnetic resonance studies that the center Figure 1 or Figure 2 would have two effects: (1) four base-pairs of a DNA Holliday junction are

3' 3'

G C=G Elongation, G C-G Next tRNA s' G C.G A ~.C A G-C pairs with Leu47----f~U-A G=C u A - U Translocation, u A. U A.U A-U S' U xu. ^ Strand exchange .. s, u u. A message "--"~'A = U U=A 4S_.,/,. U = A A.U Dr 4~ #...U = A A-U I= 46 E-U=A A=U G¥ C.G G.C ~ G,lY---" C-G G-C "--X..c.Q ~-u ] ) / "-X-C-Q~G.U ] /Gly~c "-~c-~vG.u = G G=C ) ) ~_,~A-U U-A/,,'-" ~ 3' U"~U.^/f ~ ~s ~. UAU,A/f---" /~p U-A u.^ ~-.- /" A U.A ~,._ /r ^ U-A. ~,, --~c.G A.U~,,J ~ A-U\ ) G A- U ~,,~ 3" , U.A-- - U-A~ U-A

Io) (b) Ic) Figure 2. Branch migration mechanism of translation in the region of the interruption. The large Figures show the secondary structures at each step. The smaller Figures schematize the relationship between these structures and a standard Holliday junction. Dotted lines show where the rest of the strand containing the tRNA anticodon loops would be if this strand were longer; the dot in the rightmost strand, marks the nucleotide across from the gap in the template shown in (a) and serves as a point of reference; shaded boxes, tRNA anticodon loops; -- and -, base-pairs. (a) Translation occurs normally until Gly46 enters the A site. (b)Either before or after aminoacylation and translocation, the branch point between the 2 helices migrates leaving the 5' end of the anticodon loop paired against a continuous template. (c) tRNA L'u pairs with the now continuous message. Communications 201 stacked onto their respective helices (Wemmer el al., (Thompson & Hearst, 1983)). This is the first 1985), it is not known whether a discontinuity in the suggestion that RNA branch migration may be mRNA template would inhibit the addition of the involved in an RNA-mediated activity. Whereas next tRNA. DNA branch migration is a spontaneous process It is not clear what the minimum structural that occurs rapidly in solution (Thompson el al., requirements for long-range translational skipping 1976), the environmental context of the ribosome might be, since this is the first such case to be could either facilitate or hinder migration of the reported. Consequently, there is no theoretical basis mRNA-tRNA complex. It will be interesting to see for predicting a translational skip from sequence the effects of altering the sequence of the inter- information. The ability to branch-migrate removes ruption in order to delineate the respective roles of the need for tRNA addition across such a discon- structure and dynamic motions of the message in tinuity, while the requirement for the pentanucleo- facilitating the translational leap. tide duplication (Weiss et al. unpublished results) is consistent with a model involving strand exchange The authors thank Larry Gold for thoughtful and care- between the dumbbell and the message. ful consideration of the manuscript. We also thank Other factors may also contribute to translation Robert Weiss and Ignacio Tinoco for useful discussions across the interruption. The very fact that the inter- and for sharing data prior to publication. This work was supported in part by NIH grant no. GM30786 and ruption can fold as a dumbbell rather than as a by the Department of Energy under contract stem-loop, the size and shape of the dumbbell, or DE-ACO30-76F00098. the presence of an unpaired third loop near the center of the dumbbell, may also play a role. This References extra loop, for example, contains an eight-nucleo- Huang, W. M., Ao, S.-Z., Casjens, S., Orlandi, R., Zeikus, tide sequence that is complementary to the ribo- R., Weiss, R., Winge, D. & Fang, M. (1988). Science, some binding sequence at the 5' end of the gene 239, 1005-1012. (Huang et al., 1988), and this overlaps a five-nucleo- Kolter, R. & Yanofsky, C. (1982). Annu. Rev. Genet. 16, tide sequence complementary to the message 113-134. immediately 3' to the dumbbell. A pseudoknot Michel, F., Hanna, M., Green, D. R., Bartel, D.P. & formed by pairing these latter two regions could 8zostak, J.W. (1989). Nature (London), 342, potentially serve to orient the dumbbell colinearly 391-395. to the rest of the message in order to allow the Price, J. V. & Cech, T. R. (1988). Genes Develop. 2, message to pass through tight constrictions on the 1439-1447. ribosome. The above model does not address the Thompson, B. J., Camien, M. N. & Warner, R. C. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 2299. question of possible contributions of this loop. Thompson, J. F. & Hearst, J. E. (1983). Cell, 32, RNA is dynamic, capable of forming multiple 1355-1365. structures from the same sequence (e.g. attenuation Wemmer, D. E., Wand, A. J., Seeman, N. C. & (Kolter & Yanofsky, 1982), self splicing (Price & Kallenbach, N.R. (1985). Biochemistry, 24, Ceeh, 1988), and ribosome conformational changes 5745-5748.

Edited by P. yon Hippel