Molecular modeling to predict the structural and biological effects of mutations in a highly conserved histone mRNA loop sequence.

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Molecular Modeling to Predict the Structural and Biological Effects of Mutations in a Highly Conserved Histone mRNA Loop Sequence a

a

Henry A. Gabb , Michael E. Harris , Niranjan B. b

b

Pandey , William F. Marzluff & Stephen C. Harvey a a

Department of Biochemistry , Schools of Medicine and Dentistry University of Alabama at Birmingham , Birmingham , AL , 35294 b

Department of Chemistry , Florida State University , Tallahassee , FL , 35205 Published online: 21 May 2012.

To cite this article: Henry A. Gabb , Michael E. Harris , Niranjan B. Pandey , William F. Marzluff & Stephen C. Harvey (1992) Molecular Modeling to Predict the Structural and Biological Effects of Mutations in a Highly Conserved Histone mRNA Loop Sequence, Journal of Biomolecular Structure and Dynamics, 9:6, 1119-1130, DOI: 10.1080/07391102.1992.10507983 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10507983

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Journal ofBiomolecular Structure & Dynamics, ISSN 0739-1102 Volume 9, Issue Number 6 (1992), "'Adenine Press (1992).

Molecular Modeling to Predict the Structural and Biological Effects of Mutations in a Highly Conserved Histone mRNA Loop Sequence Henry A. Gabb\ Michael E. Harris\ Niranjan B. Pandey2, William F. Marzluf:F and Stephen C. Harvey1•* Downloaded by [Rutgers University] at 17:04 07 April 2015

1

Department of Biochemistry Schools of Medicine and Dentistry University of Alabama at Birmingham Birmingham, AL 35294 2

Department of Chemistry Florida State University Tallahassee, FL 35205

Abstract The 3' -end of histone mRNAs contains a highly conserved sequence motif which is believed to form a 6 base pair stem and a 4 base loop. These sequences are involved in both the efficiency of 3' -end formation and stability of the mature histone mRNA We have modeled four stem basepairs and the loop portion of this structure using the wildtype sequences and several mutant sequences. A structure for the wildtype stem-loop is proposed that is based on energy minimization using a representative wildtype sequence and comparison with structures obtained using naturally occuring mutations which do not alter loop function. A wildtype structure is proposed in which the top basepair of the stem is broken, forming a six base loop. Mutant sequences with altered bases in the loop and in the stem were also modeled. The effect of these mutations on the proposed wildtype structure is discussed and possible biological consequences considered.

Introduction The steady state amount of mRNAfrom replication-dependent histone genes varies during the cell cycle. Regulation occurs at multiple levels including transcription, efficiency of 3'-end formation, and mRNA stability (reviewed in 1,2). Histone mRNAs are not polyadenylated but rather end in a conserved sequence, 5' -GGYYYUUYUNARRRCC3', which is believed to form a stem-loop structure (3,4). The sequences which form this stem-loop are important for regulation of 3' -end formation as well as mRNA stability(5-8). These conserved sequences are generally believed to form a six base pair stem and a four base loop (Figure 1). *Author to whom correspondence should be addressed.

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Gabb etal.


1120

G

G

5'

c c

3

I

Figure 1: Proposed secondary structure for the conserved sequence located at the 3' terminus of replication-dependent histone mRNAs. Y and R denote conserved pyrimidines and purines, respectively. N represents a non-conserved base in the sequence. The bases above the horizontal line were modeled in our study.

We have used molecular modeling to determine a possible three dimensional structure for the histone mRNA 3' stem-loop. A structure is proposed based on energy minimization using the wild type sequence and comparison with structures obtained using naturally occuring mutations which do not alter loop function in vivo. Interestingly, the absolutely conserved U4-A9 basepair (Figure 1) is broken in our lowest energy wild type structures resulting in a loop containing six bases rather than four. This is expected since experiments on RNA hairpin loops with A-U bp' sat the top of the stem suggest that 6-membered loops are more stable than 4-membered loops (9). RNA hairpin loops with G-C bp' s at the top of the stem, however, show a preference for 4-membered loops over 6-membered loops (1 0). Also, the bases in the loop itself assume a particular stacking pattern. One position in the loop, N8 (Figure 1), is not conserved in nature. The four structures containing alterations at this position were essentially superimposable after energy minimization. This is to be expected since 3'-end formation occurs normally in vivo regardless of the base at position 8 (1,2). Mutations in conserved positions of the stem as well as mutations in the loop were modeled. These mutations are currently being tested in vivo (Marzluff and coworkers, manuscript in preparation). We found that some mutations altered either loop size or loop geometry, while others had little effect on structure. Predictions are made concerning possible biological effects of these mutations on loop function or recognition.

Systems and Methods

Loop Generation All loops were generated as described previously (11), using the fiber diffraction


Mutation in RNA Loops

1121

A Figure 2: Generation ofloop structures. A. Starting A-type RNA helix of sequence (5'-GCCCUAAAAA3' · 5'-UUUUUAGGGC-3'). B. Unrefined loop in 404 configuration of sequence (5'-CCCU-3' · 5'UUUUAGGG-3'). Note that the single stranded region is in ideal A-helix configuration. C. Final loop of sequence 5'-CCCUUUUUAGGG-3' minimized with JUMNA

coordinates of an A-helix as a starting point. A template A-type RNA double helix was constructed with RJMNA3e (Junction Minimization of Nucleic Acids), a molecular mechanics package for building and refining macromolecular models (12). Next, the CURVES 3.0 program (13) was used to remove unnecessary nucleotides from the template helix and close the loop. Finally, RJMNA was used to minimize the unrefined loop to convergence without constraints (Figure 2). An Iris 40/220 GTX was used for all calculations and the resulting structures were displayed on a Silicon Graphics console using the Quanta/CHARMm molecular modeling package (available from the Polygen Corporation). RJMNA output files were converted to Protein Data Bank format with the program MACGENPDB (written by T. You, UAB, Dept. of Physics). All test sequences were modeled as four-membered loops with the U4-A9 base pair intact before minimization. The sequences modeled are listed in Tables I and II. Loop configurations are described according to theN = L + M system ( 11 ), where N is the number of bases in the loop, Lis the number ofbases stacked on the 5' strand, and M is the number of bases stacked on the 3' strand. All five permutations of a four-membered loop (L=O,l, ... ,N) were examined for each sequence. For a given sequence, the permutation with the lowest energy is the favored conformation ( 11 ). Since potential energies are measured relative to an arbitrary zero point the absolute energy of a given structure is not as important as the difference in energies between structures of the same series (14). Consequently, potential energies within a given series are suitable for comparison while energies between different loop sequences are not directly comparable. Solution and Ion Effects

Since a twelve nucleotide system is too large for inclusion of explicit solvent molecules

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during minimization, artificial solvent methods were used for these studies. JUMNA has two built-in methods to simulate solvent conditions. First, ion screening is simulated by reducing the total charge on the phosphate groups. Phosphate charges were set to -0.5 during minimization. Second, the Hingerty dielectric damping model (15) is included in the JUMNA potential function. The Lee and Richards algorithm (16) (adapted to acceptJUMNAoutput files byT. You, UAB, Dept. of Physics) was used to calculate the solvent accessible surface of the structures minimized by JUMNA This algorithm determines the solvent exposed surface area of a structure by rolling a sphere over its surface. For our calculations, a probe sphere of radius 1.4 A(the generally accepted radius of water) was used. Solvation energy was estimated by multiplying the surface area by a generally accepted conversion factor of 25 cal/mol· N obtained from solvent transfer experiments (17). It should be noted that other experimental methods place this value much higher(l8,19). More recent data, however, suggests that lower values are more correct (20). The potential energy determined by JUMNA was added to the solvation energy to give the final energy of the structure. A MicroVax 2000 was used for the surface area calculations. Table I Wildtype Sequences and Summary of Results Lowest Energy Starting Geometry

P.E.

P.E.+S.E.

Minimized Loop Geometry

1. CCCUUUUUAGGG

404* 413 422 431 440*

-10.1 14.4 31.1 1.5 -13.8

20.4* 41.9 58.3 29.1 15.8*

615/624 NDG4-1oop NDG4-Ioop NDG4-1oop NDG4-Ioop

2. CCCUUUUCAGGG

404 413 422 431 440

-20.1 -3.6 30.4 6.1 -15.3

11.6 21.7 57.3 34.8 12.7

615/624 NDG 6-1oop NDG4-1oop NDG4-Ioop 651

3. CCCUUUUAAGGG

404 413 422 431 440

-18.6 13.8 18.6 6.3 -2.4

11.3 40.3 45.2 36.4 25.9

615/624 NDG4-loop NDG6-Ioop 211 642

4. CCCUUUUGAGGG

404 413 422 431 440

-10.2 10.7 3.7 -1.7 -5.9

21.6 41.2 33.6 26.9 24.7

615/624 413 NDG4-loop NDG4-loop 651

Sequence

Energies are given in Kcal/mol. The lowest energy geometry for each sequence is underlined. P.E.= potential energy calculated by JUMNA S.E.= solvation energy. N.D.G.= no definable geometry. * See text for discussion.


1123

Results and Discussion


Selection of the Wildtype Structure

An important part ofthis study is the proper selection of a starting structure to serve as a basis of comparison for the mutations being tested. Table I shows the sequences used in the wild type test group. Examination of the lowest energy structures from the first four sequences revealed certain recurring features. First, the U4-A9 base pair is always broken after minimization consistent with thermodynamic experiments on hairpin loops with A-U bp's at the top of the stem (9). Second, the minimized loops show a 615/624 geometry. This is consistent with Hasnoot's rules governing loop size and stacking pattern for nucleic acids (25) and similar to the known structures oftRNA anticodon loops (21,22). Third, the hydrogen bonding positions of the 5'-stacked bases (position 5 in particular) are facing the solvent. Fourth, the base at position 4 always lies in the major groove of the stem. A representative wildtype loop is shown in Figure 3. These loops also share a common pattern of hydrogen bonds. They all have an OH2':04' hydrogen bond between residues 7 and 8 or residues 8 and 9. Also, uracil-4 and the base at position 8 are always partially base paired. In fact, when guanine occupies position 8 a wobble base pair forms. The lowest energy structure for 5' -CCCUUUUUAGGG-3' was not chosen. The 440 structure was disqualified because its geometry was not consistent with Hasnoot' s rules (which predict extensive 3'-stacking) (25), crystal data oftRNAloops (21,22), or NMR studies of loops (23,24). Also, it showed no similarity with the other lowest energy wildtype structures. Instead, the next lowest energy structure was chosen since it displayed the characteristics outlined above. Analysis of Mutant Sequences

Since we are attempting to give structural reasons for the effects of mutations in the

0 0

u

0 0

A C- G C- G C- G

5'

3'

Figure 3: Stereoimage of a representative wildtype loop structure. The sequence shownjs 5'-CCCUUUUUAGGG-3'. The scale in the upper right corner of each image is in Angstroms.

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Gabb eta/. Table II Mutant Sequences and Summary of Results

Mutation


I. 2. 3. 4. 5. 6. 7. 8. 9.

Lowest Energy Starting Geometry

P.E.

P.E.+S.E.

440 440 404 440 440 440 440 404 404

-41.0 -46.1 -19.8 -11.7 -17.3 -22.0 -ll.8 -24.6 -20.5

-11.2 -15.8 11.7 15.5 9.8 7.8 16.5

(U4-+C, A9-+G) (U4-+G, A9-+C) (A9-+G) (U5-+G) (U5-+A, U7-+A) (U5-+G, U7-+G) (Cl-3,U4-+G,A;Gl0-12,A9-+C,U) (U7-+A) (U4-+A, A9-+U)

3.6

10.3

Minimized Loop Structure 4-loop 4-loop 4-loop 651 NDG6-loop NDG 6-loop 642 615 615

Energies are given in Kcal/mol. P.E.= potential energy calculated by JUMNA S.E.= solvation energy. N.D.G.= no definable geometry.

highly conserved 3'-end of histone mRNA, we next obtained minimized structures for several mutant loop sequences. Some of the mutations described below have been tested by an in vivo assay (Marzluff and coworkers; manuscript in preparation). Some, however, have not yet been tested (Marzluff and coworkers; work in progress). Predictions of the biological effects of these mutations are included to test the accuracy of our modeling data. Table II shows all of the mutations tested in this study. The mutant structures are grouped into three categories: mutations affecting loop size, mutations affecting loop geometry, and those that do not appreciably affect loop size or geometry. Mutations Affecting Loop Size Initially, we assumed that the wildtype structure contained a four-membered loop because of the complementary bases at the top of the stem (Figure 1). Surprisingly, however, minimization causes this bp to break in all of our wildtype structures (Figure 3). We concluded that mutations causing a change in loop size would have drastic effects on the regulation of histone mRNA concentration. (U4-C. A9-G) and (U4-G, A9-C): The extra G-C bp stabilizes the stem and gives a 4-loop rather than a 6-loop (Figure 4a,b ). This is in agreement with thermodynamic data showing a preference for4-loops over6-loops when there is a G-C bp at the top of the stem (10). The sugar-phosphate backbones of these two loops are nearly superimposable whereas the base orientations in the loops are different. There is extensive base-backbone and backbone-backbone hydrogen bonding but no stacking in the loop. (A9-G): This mutation caused a U4-G9 wobble base pair to form at the top of the stem (Figure 4c). This is somewhat surprising since this type ofbp has a lower melting temperature than a standard Watson-Crick U-A base pair (26).


1125

A u

0

u

0

G C- G


C -

c:.G c-G 5'

3'

8 u

uu

G -

u C

C - G C - G C - G

s•

3'

c u u

u

u

U- G C - G

C - G C - G

s•

3'

Figure 4: Stereoimages of the mutations affecting loop size. A (U4-+C, A9-+G ). B. (U4-+G, A9-+C). C. (A9-+G) Note that a wobble base-pair is formed at the top of the stem. The scale in the upper right corner of each image is in Angstroms.

Gabb etal.

1126

A uu

c:

U

u

A

c- c c- c c- c

s•

l'


8

U&

.. V

u A

c-c c-c c-c

5'

l'

c uc:

c: 0

u

A

c- c c - c c- c s• J'

D u 0 ..

0

u 0

c: - c G- C

G - C 5' ]•

Figure 5: Stereoimages of mutations affecting loop geometry. A. (U5-G). B. (U5-A, U7-+A). C. (U5-G, U7-+G). D. (Cl-3,U4-+GA Gl0-12,A9-+C,U). The scale in the upper right corner of each image is in Angstroms.


1'127

Mutations Affecting Loop Geometry


(U5-G): This mutation gave a final minimized geometry of 651 (Figure Sa). Positions 6-8 are facing the solvent and the pattern of base-backbone and backbone-backbone hydrogen bonding is similar to that ofthe wildtype. However, the stacking geometry is opposite that of the wild type and the base at position 9 is lying in the major groove of the stem rather than the base at position 4. (U5-A, U7-A) and (U5-G, U7-G): These two struct]Jres (Figure Sb,c) are very similar. Neither one has a categorizable stacking pattern and positions 7 and 8 are facing the solvent Also, both show extensive base-backbone and backbone-backbone hydrogen bonding. A double pyrimidine to purine mutation in a loop is expected to affect that loop's structure because of the added size of the purine bases. (Cl-3,U4-G,A; GJ0-12,A9-C,U): Reversing the pyrimidine/purine strands in the stem has far-reaching effects on the loop structure (Figure Sd). This mutation adopts a 642 geometry after minimization. There is no base-backbone hydrogen bonding but uracil-5 is hydrogen bonded to uracil-9. Since the pyrimidine/purine positions in the stem are strictly conserved (Figure 1) we suggest that the pattern of hydrogen bond donors and acceptors in the major and minor grooves is important for protein binding in a manner similar to restriction enzyme recognition in DNA

A 11 A 11

11 11

A

C- G C- G C- G

s•

J'

8 11 11

u A

u V

C- G C- G C- G

s•

J•

Figure 6: Stereoimages of mutations that do not appreciably affect loop geometry. A. (U7-+A). B. (U4--+A, A9-+U). The scale in the upper right comer of each image is in Angstroms.

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Gabb eta/.

Mutations Affecting Neither Loop Size Nor Geometry The structures of this class closely resemble the wildtype suggesting that alteration of3' -end formation and steady-state concentration of histone mRNA would be due to subtle interactions with the binding site of the protein that escorts the mRNA from the nucleus to the cytoplasm.


(U7-A): This structure (Figure 6a) is similar to the wildtype loop (Figure 3). Since

position 7 is a strictly conserved uridine, mutation to a purine is expected to affect proper functioning of the loop in vivo. If this is the case, then we can attribute the effect to two things: 1. steric problems caused by the larger base and 2. a reversal of the hydrogen bond donor/acceptor positions. The latter problem could be corrected by rotating the glycosidic bond of the purine from anti to syn. If the location of the hydrogen bonding positions of the uracil-7 are important for protein binding, then a U7-C mutation would be detrimental since the energy barrier for an anti to syn glycosidic transition is prohibitively high for pyrimidines (27). (U4-A, A9-U): This structure (Figure 6b), though similar to the wildtype, shows

some differences. Even though it is still a 6-loop, there is no longer a definite stacking pattern. However, since these positions are strictly conserved we can assume that they are somehow involved in protein binding though this is unclear at present because very little is known about the protein(s) involved.

Conclusions We suggest that loop size and geometry are very important for proper 3' -end formation in histone mRNA These modeling studies give information on three aspects of the mRNA stem-loop structure: loop size, stacking patterns within the loop, and nucleotide positions which may be necessary for protein binding. In addition, experimentally testable predictions are made about the consequences of mutations at highly conserved sites within the stem and loop. For the wildtype sequences, six-membered loops are more stable than four-membered loops. This is only possible ifthe bases atthe top of the stem, U4 and A9, are not base paired (Figure 3). With regard to stacking patterns within the loop, ourlowest energy wildtype structures are 3'-stacked, with 615/624 final geometries. This is consistent with stacking patterns in other RNA loops, as determined by X-ray crystallography (21,22), NMR(23,24), and theoretical studies (25). Phylogenetically, wildtype sequences allow any nucleotide at position 8; the four model wildtype loops (containing A, C, G, and U at position 8) all have similar structures, indicating that loop geometry is insensitive to sequence at that position. We would predict that mutations stabilizing the top of the stem, producing fourmembered loops, would disrupt the structure and alter biological activity. Consequently, the introduction of a G-C bp at the top of the stem should reduce or eliminate regulation of3'-end formation. Surprisingly, when we introduced a Gat position 9, an intact wobble bp formed with uracil-4, so we predictthatthis mutation


1129


would also affect regulation of 3'-end formation. We believe that deleterious mutations within the loop fall into two classes. First, those mutations that clearly change the loop structure may affect regulation by an unknown mechanism. Second, those mutations which do not appear to affect loop geometry in the modeling studies but may disrupt 3'end formation by changing the subtle electrostatic interactions with the necessary protein active site(s). Because the loops built by unconstrained minimization are so similar in structure to RNA loop structures determined by X-ray crystallography (21,22) and NMRIdistance geometry methods (23,24) and follow thermodynamic data on loop size (9,10), we are confident in our loop building protocol, the JUMNA potential function, and our structure-based predictions for the biological effects of mutations in the 3'-end of histone mRNA

Acknowledgments We are indebted to Dr. Richard Lavery for kindly making JUMNA and CURVES available to us, Dr. Steve Hajduk for helpful discussions, Dr. Arun Malhotra for assistance with QUANTNCHARMm, and to Mr. Alex McBride for his hospitality during the preparation of this manuscript. This work was supported by a grant to S.C.H. from the National Institutes of Health (GM-34015). References and Footnotes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Schumperli, D., Cell45, 571 (1986). Marz1uff, W.F. and Pandey, N.B., Trends Biochem. Sci. 13,49 (1988). Bimsteil, M.L., Busslinger, M. and Strub, K., Cell41, 349 (1985). Hentschel, C.C. and Bimsteil, M.L., Cell25, 301 (1981). Levine, BJ~ Chodchoy, N~ Marzlutt W.F. and Skoultchi, A.L Proc. Nat/. Acod. Sci US.4 84, 6189 (1987). Mowry, K.L. and Steitz, J.A, Mol. Cell. Bioi. 7, 1663 (1987). Stauber, C., LUscher, R, Eckner, R., LOtscher, E. and SchUmperli, D., EMBO J. 5, 3297 (1986). Liu, T.-J., Levine, B.J., Skoultchi, AI. and Marzluff, W.F., Mol. Cell. Bioi. 9, 3499 (1989). Uhlenbeck, O.C., Borer, P.N., Dengler, B. and Tinoco, I., Jr.,J. Mol. Bioi. 73,483 (1973). Groebe, D.R and Uhlenbeck, O.C., Nucleic Acids Res. 16, 11725 ( 1988). Harvey, S.C., Luo, J. and Lavery, R.,Nucleic Acids Res. 16, 11795 (1988). Lavery, R in Unusual DNA Structures, RD. Wells and S.C. Harvey, (eds.), Springer-Verlag, New York, pp 189 (1987). Lavery, Rand Sklenar, H.,J. Biomol. Struct. Dynam. 6, 63 (1988). McCammon, J.A and Harvey, S.C., Dynamics ofProteins and Nucleic Acids, Cambridge University Press, 1987. Hingerty, B., Richie, RH., Ferrel, T.L. and Turner, I.E.,Biopolymers 24,427 (1985). Lee, B. and Richards, F.M.,J. Mol. Bioi. 124,523 (1971). Chothia, C., Nature 248,338 (1974). Shortie, D., Stites, W.E. and Meeker, AK., Biochemistry 29, 8033 (1990). Sharp, K.A, Nicholls, A, Fine, R.F. and Honig. B., Science 252, 106 (1991). Eriksson, AE., Baase, W.A, Zhang. X-J., Heinz, D.W., Blaber, M., Baldwin, E.P. and Matthews, B.W., Science 255, 178 (1992). Sussman, J.L. and Kim, S.-H., Science 192,853 (1976). Moras, D., Comarmond, M.B., Fischer, J., Weiss, R., Thierry, J. C., Ebel, J.P. and Giege, R,Nature 288,669 (1988). Cheong, C., Varani, G. and Tinoco, I., Jr., Nature 346, 680 (1990). Heus, H.A and Pardi, A, Science 253, 191 (1991). Haasnoot, C.AG., Hilbers, C.W., van der MareI. G .A, van Boom, J.H., Singh, U.C., Pattabiraman, N. and Kollman, P.AI.,Biomol. Struct. Dynam. 3, 843 (1986).

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Date Received: October 24, 1991

Communicated by the Editor R.H. Sarma

Note Added in Proof


William F. Marzluff, Present address: Program in Molecular Biology, University of North Carolina, Chapel Hill, NC 27599.

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