[1] Determination of a transfer RNA structure by crystallographic method

[1]

CRYSTALLOGRAPHIC

DETERMINATION

OF tRNA

[1] D e t e r m i n a t i o n o f a T r a n s f e r R N A S t r u c t u r e Crystallographic Method

By

3

STRUCTURE

by

S U N G - H O U K I M a n d GARY J . QUIGLEY

Unlike most biochemical techniques, the determination of the threedimensional structure of a macromolecule by X-ray crystallography requires a long time to learn and to perform, primarily because the physical theory and mathematics of diffraction analysis are quite complex. However, it is not difficult to understand the method conceptually. This chapter is therefore intended to describe the procedures involved in the determination of a crystal structure of a tRNA to interested biochemists, without delving too deeply into the technical or mathematical details (for a relatively simple introduction, see Holmes and Blow1). These procedures can be considered as a guide to workers wishing to investigate other polynucleotide structures, although similar methods initially and repeatedly have been used to solve protein structure. Determination of the crystal structure of a transfer RNA involves four principal steps: (a) crystallization of the molecule, (b) collection of X-ray diffraction data from "native" and "heavy-atom derivative" crystals, (c) construction and interpretation of electron-density maps, and (d) refinement of the crystal structure. Of these four steps, the art of crystallization is the least understood but probably the most important; indeed, it is often the "rate-limiting" step in structural determination. Because there is no one general method for obtaining single crystals suitable for X-ray diffraction, we will describe in the next section how various transfer RNAs have been crystallized and the quality of those crystals where known. Technical details, density measurement of crystals, and crystal mounting are also described. In a third section we use an optical analogy to describe X-ray diffraction phenomena and the "multiple isomorphous replacement" method and also describe the construction and refinement of molecular models from the diffraction data. A fourth section summarizes the structural features of transfer RNA that may be considered reliably determined and functionally significant.

i K. C. Holmes and D. M. Blow, "The Use of X-Ray Diffraction in the Study of Protein and Nucleic Acid Structure." Wiley, New York, 1966. METHODS IN ENZYMOLOGY, VOL. LIX

Copyright © 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181959-0

4

TRANSFER RNA AND AMINOACYL-tRNA SYNTHETASES

[1]

Crystal Preparation Growing Crystals

The growing of crystals of biological macromolecules suitable for Xray diffraction is a poorly understood process and depends heavily on an empirical approach, i.e., trying many different conditions for a given molecule. A general review of this topic is given by McPherson.2 The most commonly used techniques are the introduction o f a precipitating agent into the macromolecular solution through either vapor-phase or liquid-phase diffusion, depending whether or not the precipitant is volatile. A vapor-diffusion method is described below as an example. Because each crystallization sample in this procedure is small (20-100 fd), the depression plates in which samples are stored are coated with a nonwetting agent, to prevent drop spreading, as follows: The depression plates are soaked in a cleaning solution for 1 hr, rinsed thoroughly with water and then with deionized water, immersed in a warm silane solution (1% dimethyldichlorosilane in benzene or toluene) for about 6 min under a well-ventilated hood, and then dried in an oven. In the vapor-diffusion method, a small drop of sample is placed in each concavity of a depression plate prepared as above. The plate rests on an inverted petri dish c o v e r inside a clear plastic box (see Fig. 1), and a solution of a given concentration of the volatile precipitant is placed in the bottom piece of the box as a reservoir. The edge of the box lid is sealed with vacuum grease and the progress of crystallization can be observed without opening the box. The concentration o f precipitant in the reservoir can be increased every 2 days or so until crystallization occurs. Similarly, crystals may be redissolved by diluting the reservoir. When nonvolatile precipitants are used, a small amount of precipitant is first introduced in the sample drop to prevent it from drying up com-

FIG. 1. One of the commonly used crystallization setups for the vapor diffusion method. (A) Clear plastic box bottom; (B) top of a petri dish; (C) depression plate containing nine concaves; (D) clear plastic box top; (E) assembled setup. 2 A. McPherson, Methods Biochem. Anal. 23, 249 (1976).

[1]

CRYSTALLOGRAPHIC DETERMINATION OF

tRNA

STRUCTURE

5

pletely, and a certain concentration of precipitant is placed in the reservoir. In this case, water from the sample drop is gradually diffused into the reservoir, thus increasing the relative concentrations of precipitant and macromolecules in the sample. The most commonly used liquid-diffusion method is the dialysis of the sample solution against the buffer solution containing a gradually varying amount of precipitant, such as ammonium sulfate, ammonium acetate, 2-methylpentane-2,4-diol, or dioxane. Because the crystallization conditions vary widely for different molecules, with no one optimum technique apparent, we summarize in Table I the conditions that have yielded tRNA crystals of different qualities. We emphasize that these conditions are to be considered only as starting guides for those wishing to make crystals of nucleic acids. Indeed, two persons using identical conditions may frequently obtain crystals of very different qualities.

Density Measurement When suitable crystals are formed, their density must be measured to determine the number of molecules per unit cell, the basic repeating unit in the crystal. This is usually done by use of a density gradient. A procedure used for yeast tRNA Phe crystals is as follows: Water-saturated m-xylene (density -0.87 g/ml) is layered gently upon water-saturated bromobenzene (density -1.52 g/ml) in a thin graduated cylinder, and the two liquids are partially mixed gently by a few vertical motions with a glass rod. The mixture is allowed to stand for several hours to form a density gradient, which is calibrated by observing the equilibrium positions attained by a number of small drops (10/zl or less) of CsC1 solutions of various densities. These reference solution densities may be measured either directly by weighing or by measuring their refractive indexes; for CsCI solutions, refractive indexes of 1.3400, 1.3600, 1.3750, 1.3900, and 1.4100 correspond to densities of 1.068, 1.274, 1.434, 1.596, and 1.816 g/ml, respectively (see Fig. 2). Finally, the density of the crystals may be obtained by dropping them into the density gradient and interpolating from the positions of tile two reference drops bracketing the crystals.

Mounting Crystals Most macromolecular crystals are unstable when exposed to air and so must be sealed in capillaries with their mother liquor (solution in which they have been grown) prior to X irradiation. In general, the thinwalled quartz or glass capillaries must be cleaned thoroughly, as described below.

6

TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES

[1]

TABLE I. CONDITIONS THAT

Source

Amino acid specificity

Initial tRNA concentration (mg/ml)

Yeast

Phenylalanine

4

Yeast

Phenylalanine

15

Yeast

Phenylalanine

15

Yeast Yeast

Phenylalanine Phenylalanine

10 10

Yeast

Phenylalanine

3.4

Yeast

Phenylalanine

~2

Yeast

Phenylalanine

10

Escherichia coli E. coli

Phenylalanine

4.5

Phenylalanine

5

E. coli

Formylmethionine

3.4

E. coli

Formylmethionine

~20

Yeast

Formylmethionine

4-10

Yeast

Aspartic acid

3

Yeast

Aspartic acid

6

Yeast

Mixture

100

Buffer

pH

Other a

Na cacodylate, 6.0 MgCIz, 10 mM; 10 mM spermine 4HCI, 1 mM Na cacodylate, 6.0 MgCI~, 40 mM; 40 mM spermine 4HCI, 4 mM Na cacodylate, 6.0 MgClz, 40 raM; 40 mM spermine 4HCI, 4 mM; EDTA, 40 mM MgSO4, 10 mM MgSO4, 20 mM, MgAc2, 20 mM Na cacodylate, 6.0 MgCI2, 10 mM 10 mM spermine, 4HCI, lmM K cacodylate, 7.0 MgCI:, 5-15 mM 10 mM spermine 4HCI, 1-3 rnM Mg 2+, 10 raM; spermine, 10 mM Tris, 5 mM 7.4 MgCI2, 5 mM Na cacodylate, 16.0 MgCI2, 10 mM; mM NaC1, 2 mM; spermine, 1 mM Tris, 5 mM 7.0 MgClz, 1 mM; MnClz, 1 mM Tris, 10 mM 7.0 MgC12, 12 mM; NaCI, 500 mM; Na~S~O3, 1 mM Na cacodylate, 5 mM Na cacodylate, 10 mM Na cacodylate, 15 mM Na cacodylate, 10 mM

6.0 NH4CI, 50 raM; MgClz, 5 mM 6.0 MgC12, 10 mM; spermine, 3 mM 6.8 MgC12, 15 mM; spermine, 3 mM 7.0 KCI, 150 mM; MgCI2, 10 mM; EDTA, 0.1 mM

[1]

7

CRYSTALLOGRAPHIC DETERMINATION OF t R N A STRUCTURE

HAVE YIELDED tRNA CRYSTALS

Crystallization method b

Temp. (°C)

Isopropanol, 10% (v/v)

VD

4

3 Days ~ P2,22, 2 weeks

33, 56, 161

Isopropanol, 8% (v/v)

VD

4

1 Day - 2 weeks

P2,221

33, 56, 161

Isopropanol, 9% (v/v)

VD

4

2 Days ~ months

14132

154

d

Dioxane, 35% (v/v) 2-Methylpentane2,4-diol (MPD) MPD, 10-13% (v/v)

VD DA 7

61, 85, 234 61, 85,234, 124, (61 °) 33, 56, 63 (a = 90 ° )

e e

VD

C2221 C222, R32 ~2 Weeks P2,

Dioxane, 10-20% (v/v)

VD

4

~3 days

P2,

33, 56, 63 (a = 90 °)

MPD, 33%(v/v)

DA

22

P6~22

107, 107, 117

h

Ethanol, 20% (v/v)

VD

1

P6222

124, 124, 160

i

Dioxane, -20% (v/v)

VD

P3112

93, 93, 78

j

Ethanol, 7% (v/v)

VD

4

C222

63, 107, ~09

k

Cetyltrimethylammonium bromide (CTA-BR or CI) 12raM (NH4)~SO4, 50% saturation Isopropanol

VD (NaCI)

23

Weeks

C222

82, 123, 110

l

VD

Weeks

P6222

115, 115, 137

m

--

Weeks

P6~22 C222, C222~

98, 98, 150 171, 98, 150 61, 98, 148

n

(NHa)2SO4, 62% saturation Dioxane, 50% (v/v)

8 22 4 20 20

VD

32

- 7 Weeks P2221

45, 52, 128

o

Precipitent

VD

Period

2 Weeks

Space group

Cell dimension (k~)

References

f

n

(continued)

8

[1]

TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES

TABLE I

Source

Amino acid specificity

Initial tRNA concentration (mg/ml)

E. coli

Tyrosine

5

E. coli

Leucine

4-10

E. coli

Glutamine

0.15

Buffer

pH

Other a

Na cacodylate

6- MgCI2, 10-18 mM; 8 spermine, 0.1 mM Na cacodylate, 6.0 MgC12, 5-7 mM; 5mM NH4CI, 50 mM; spermine, 1 mM Tris, 10 mM 7.0 NaCI 500 mM; MgCI2, 12 raM; MnCI2, 2.5 mM; N~$203, I mM

a Some anions were not identified in the literature. b VD, vapor diffusion; DA, direct addition. c S.-H. Kim, G. J. Quigley, F. L. Suddath, and A. Rich, Proc. Natl. Aead. Sci. U.S.A. 68, 841 (1971). d S.-H. Kim, G. J. Quigley, F. L. Suddath, A. McPherson, D. Sneden, J. J. Kim, J. Weinzied, and A. Rich, J. Mol. Biol. 75, 421-428 (1973). e F. Cramer, F. von der Haar, K. C. Holmes, W. Saenger, E. Schlimme, and G. E. Schulz, J. Mol. Biol. 51, 523 (1970). J T. Ichikawa and M. Sundaralingam, Nature (London) New Biol., 236, 174 (1972). o j. Ladner, J. Finch, A. Klug, and B. Clark, J. Mol. Biol., 72, 99 (1972). n F. Cramer, R. Sprinzl, N. Furgaq, W. Freist, W. Saenger, P. C. Manor, M. Sprinzl, and H. Sternbach, Biochim. Biophys. Acta 349, 351 (1974). ~A. Hampel, M. Labanauskas, P. G. Connors, L. Kirkegard, U. L. RajBhandary, P. B. Sigler, and R. M. Bock, Science 162, 1384 (1968).

The sealed ends of the thin walled (10/zm) capillaries; (distributed by Charles Supper Co., Natick, Massachusetts) are cut open and soaked overnight in 6 N HC1; air bubbles are removed by gently tapping the container. They are then rinsed thorOughly with deionized water, soaked overnight in a buffer solution similar to that used in crystal growing, rinsed again in deionized water, and dried in an oven. The most delicate step in crystal preparation is that of mounting the crystals in the cleaned capillaries; this requires practice and extreme patience. The technique we used most often employs a micromanipulator in the following series of steps (see Fig. 3): 1. The dimensions of the desired crystal are measured under a microscope. 2. Two capillaries are selected, one with a diameter approximately the size to accommodate the chosen crystal and the second about twice as large.

9

CRYSTALLOGRAPHIC DETERMINATION OF tRNA STRUCTURE

[1]

(continued)

Precipitent Dioxane, 28% (v/v), or 6% (v/v) (NH4)2SO4, 35-45% saturation CTA

Crystallization method b

Temp. (°C)

VD Dialysis VD

8

VD (NaCI)

23

Period

Space group

Cell dimension (/~)

References

5-10 Days P4122

71, 71, 174

j

4 Weeks

46, 46, 139

p, q

75, 75, 195

l

P41

R. S. Brown, B. F. C. Clark, R. R. Coulson, J. T. Finch, A. Klug, and D. Rhodes, Eur. J. Biochem. 31, 130 (1972). k S.-H. Kim and A. Rich, Science 166, 1621 (1969). t A. D. Mirzabekov, D. Rhodes, J. T. Finch, A. Klug, and B. F. C. Clark, Nature (London), N e w Biol. 237, 27 (1972). m C. D. Johnson, K. Adolph, J. J. Rosa, M. D. Hall, and P. B. Sigler, Nature (London) 226, 1246 (1970). n R. Gieg6, D. Moras, and J. C. Thierry, J. Mol. Biol. 115, 91 (1977). o R. D. Blake, J. R. Fresco, and R. Langridge, Nature (London) 225, 32 (1970). P M. Labanauskas, P. G. Connors, J. D. Young, R. M. Bock, J. W. Anderegg, and W. W. Beeman, Science 166, 1530 (1969). J. D. Young, R. M. Bock, S. Nishimura, H. Ishikura, Y. Yamada, U. L. RajBhandary, M. Labanauskas, and P. G. Connors, Science 166, 1527-1528 (1969).

3. The tip of the larger capillary is cut as flush and even as possible. 4. The wide opening of the larger capillary is inserted in the rubber tubing attached to a micromanipulator and sealed with vacuum grease around the contact region. 5. The tip of the large capillary is positioned directly above the chosen crystal in the depression plate, using a micromanipulator and a microscope. 6. The crystal and some mother liquor are drawn into the capillary, using the screw knob of the micropipetter. 7. The wide opening of the smaller capillary is fit snugly over the tip of the larger capillary. 8. The crystal and mother liquor are pushed into the smaller capillary by turning the micropipetter knob until the crystal is lodged firmly. 9. Excess mother liquor is removed from the surface of the crystal by capillary action with thin strips of filter paper or with a glass fiber.

10

[1]

TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES

1.900

1.800

1.700

E

1.600

1.5oo c~ 1.400

1.300

1.201]

1.100

i

i.~4o

i

1.35o

a

~.~eo

i

I.~?o

Refractive

I

|

1.3eo L39o

i

|

L4oo

index

FIG. 2. Relationship between refractive index and density of CsC! so|ution.

10. A small column of mother liquor is left in the capillary. 11. Excess lengths of capillary are cleaved at one end and sealed with dental wax; then the same is done at the other end. 12. The capillary containing the crystal,is mounted on a goniometer. Now it is ready for X-ray exposure. X - R a y Diffraction

Imaging Imaging of an object using an optical microscope depends on the ability of the lens to bend light. When light passes through a specimen, it scatters in all directions. H o w e v e r , if the specimen is a periodic structure, the scattering (diffraction) patterns b e c o m e discrete. These diffracted rays can be recombined by a lens to form an image and magnified by another lens for viewing. The resolution of the image formed is directly related to the wavelength of the light, provided other conditions are the same, best resolution occurring when the wavelength is close to or less

[1]

CRYSTALLOGRAPHIC DETERMINATION OF t R N A STRUCTURE

11

FIG. 3. A crystal mounting setup. (A) Metal block; (B) crystal aligning adaptor on a magnet; (C) goniometer, a crystal orienting device; (D) microscope; (E) depression plate; (F) rubber tubing with a capillary at the end; (G) micromanipulator; (H) micropipetter. than the smallest distance it is necessary to see. To determine the atomic a r r a n g e m e n t within a molecule, therefore, a radiation wavelength is needed that c o r r e s p o n d s to interatomic distances (about 1 A); this corresponds to the wavelength of X rays. Unfortunately, there is no known lens for X rays, so a direct imaging cannot be achieved in this case. Crystallographers o v e r c o m e this problem through a complex and laborious computation by which the image (the electron density of the molecule) is reconstructed mathematically. Diffracted b e a m s of X rays are recorded on photographic film or by a radiation counter, and angle as well as intensity of each diffracted b e a m is measured. For large molecules, the n u m b e r of data points is usually at least several thousands. The mathematical operation analogous to the optical lens is Px = ~] Fh exp (i~bh)exp

(-27rih.x)

(1)

h

where px, the electron density at position x in the crystal, can be calculated if the diffraction angle, h, the square root of the intensity, Fh, and the phase angle, ~bh, are known for each diffracted b e a m . The diffraction angles and intensities are directly measurable, but the phase of each b e a m , which ranges from 0 ° to 360 °, cannot be directly measured.

12

TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES

[1]

Multiple Isomorphous Replacement Method Fortunately, there exists a technique, known as multiple isomorphous replacement, that allows determination of the phase for each diffracted beam of a crystal of a biological macromolecule. This method depends on the possibility of attaching heavy atoms (hence powerful X-ray scatterers) to a specific site or sites of each molecule in the crystal. These heavy-atom derivatives are normally obtained by introducing a salt of, for example, platinum, mercury, uranium, or rare earth ions into a solution containing normal, or "native," crystals. The heavy-atom derivative crystals therefore are identical to the native crystals, except for the introduction of a regular array of heavy atoms. The diffraction patterns from the two crystals are the same in all aspects except for definite, small differences in diffracted intensities (see Fig. 4). These differences allow location of the positions of the heavy atoms in the crystal, which in turn allows calculation of diffraction intensity and phase for each diffraction component from the heavy atom alone by the expression Fh c exp (i~bhc) = ~] f~ (h) exp (27rih.X~)

(2)

J

where Fh c and ~bhc are the calculated amplitude and phase of a diffracted beam coming out at an angle h because of the heavy atom j, which has X-ray scattering power fj (h) and is located at position Xj in the crystal. Then, given the amplitudes of the diffracted beams from a native crystal, a heavy-atom derivative, and the heavy atoms alone, plus the phase value for the heavy atoms, the three amplitudes can be triangulated to find two possible phase values for each diffracted beam for the native crystal. With a second heavy-atom derivative, a similar triangulation of three sets of amplitude data again produces two possible phases for each native crystal diffraction. Of these four phases, two have approximately the same value, which then corresponds to the correct phasing for one beam. This operation is repeated for each of the many thousand reflections, and the obtained phases are incorporated in Eq. (1) to calculate the electron densities at every point in the crystal matrix (for further details, see Ref. 1). The types of heavy-atom derivatives, and their soaking conditions, for yeast phenylalanine tRNA crystals are listed in Table II.

Electron-Density Map For such large molecules as proteins and tRNA, it is clear that only through extensive use of high-speed computers can the solution of so many repetitive calculations be achieved in any practical time limit. For

kkgk

k A

13

C

FIG. 4. Illustration of multiple isomorphous replacement method. (A) Native "crystal" of duck-shaped molecule and its diffraction pattern. (B) First heavy-atom derivative "crystal" and its diffraction pattern. A circular heavy " a t o m " is located at the back of each duck. (C) A second heavy-atom derivative "crystal" and its diffraction pattern. A triangular heavy " a t o m " is located at the bottom of each duck. To appreciate the small intensity differences, compare the intensities of equivalent spots in, for example, the second columns from the center of the three diffraction patterns.

14

TRANSFER RNA AND AMINOACYL-tRNA SYNTHETASES

[1]

TABLE II HEAVY-AToMDERIVATIVES,AND THEIR SOAKINGCONDITIONS,FOR YEAST PHENYLALANINEtRNA CRYSTALS

Compound

Concentration (mM)

trans-Diaminodichloroplatinate, trans-

0.2

1-2 Days

a

1.0 1.0

>2 Days -1 Week

b b

1.0 1.0

-1 Week I-2 Days

b a

1.0 0.2-0.5 1.0 0.2-0.5 1.0 1.0 KzOsO4, 2 raM; pyridine, 62 mM 1.0 Solid added Solid added

2-7 Days 2-3 Days 2-7 Days 2-3 Days 2-7 Days 2-7 Days 2 Weeks

b a b a b b c

- 2 Weeks - 2 Weeks ~2 Weeks

b b b

PtCI2(NH3)2 Potassium tetrachloroplatinate, K2PtCI4 Potassium tetracyanoplatinate, K2Pt(CN)4 Sodium aurous cyanide, NaAu(CN)z Hydroxymercurihydroquinone-O, Odiacetate Samarium acetate, Sm(CH3COO)3 Lutetium acetate, Lu(Ch3COO)3 Lutetium chloride, LuClz Praseodymium nitrate, Pr(NOz)3 Acetates of Eu, Tb, Dy, Gd Bis(pyridine) osmate, [Py]zOsO3 Potassium osmate K2OsO, Bis(pyridine) osmate-ATP Bis(pyridine) osmate complex of CTP, UTP, AMP, GMP

Soak time

Reference

a A. Jack, J. E. Ladner, and A. Klug, J. Mol. Biol. 108, 619 (1976). F. L. Suddath, G. J. Quigley, A. McPherson, D. Sneden, J. J. Kim, S. H. Kim, and A. Rich Nature (London) 248, 20 (1974) and our unpublished results. c R. W. Schevitz, M. A. Navia, D. A. Bantz, G. Cornick, J. J. Rosa, M. D. H. Rosa, and P. B. Sigler, Science 177, 429 (1972).

y e a s t p h e n y l a l a n i n e t R N A in a n o r t h o r h o m b i c c r y s t a l l i n e f o r m , the elect r o n d e n s i t i e s for o v e r 200,000 p o i n t s in a u n i q u e p o r t i o n (the a s y m m e t r i c unit) of the c r y s t a l h a v e b e e n c a l c u l a t e d u s i n g Eq. (1) a n d c o n t o u r e d a c c o r d i n g to the m a g n i t u d e s o f the e l e c t r o n d e n s i t i e s . U n l i k e e l e c t r o n d e n s i t y m a p s from small m o l e c u l e s , the m a p s f r o m large biological mole c u l e s u s u a l l y do n o t s h o w the i n d i v i d u a l a t o m s r e s o l v e d ; for e x a m p l e , in the case of this t R N A , W a t s o n - C r i c k b a s e pairs a p p e a r as s h o w n in Fig. 5. T h e s t r u c t u r e d e t e r m i n a t i o n b y X - r a y diffraction m e t h o d s at this stage t h e r e f o r e is n o t a n o b v i o u s direct r e s u l t of a n e l e c t r o n d e n s i t y m a p b u t a n i n t e r p r e t a t i o n o f the e l e c t r o n - d e n s i t y m a p . T h e i n t e r p r e t a t i o n o f the e n t i r e e l e c t r o n - d e n s i t y m a p p r o d u c e s a m o d e l for the s t r u c t u r e . A t this stage, the m o d e l is still s u b j e c t to c h a n g e .

[1]

CRYSTALLOGRAPHIC DETERMINATION OF

tRNA

STRUCTURE

15

FiG. 5. Interpretation of 2.7 ,~ resolution electron density map before structure refinement. Notice how each base, ribose, and phosphate appears at this stage as a partially separated peak.

Structure Refinement The last stage of the structure determination is the refinement of the model obtained from the electron-density map interpretation. There are two general methods of refining a crystallographic structure model. One is fitting.the model into the successively improved electron-density m a p s by minor adjustment of atomic positions. The second method is the adjustment of atomic positions so that the calculated diffracted b e a m intensities from the atomic coordinates using Eq. (2) are as close as possible to the values o b s e r v e d from X-ray diffraction experiments. At present there is no very convincingly good numerical criterion that can be depended on as a measure of the goodness of the structure determination. There are two generally used criteria: one is visual inspection of the regions where the model is fitting very poorly, as well as average fitting; the second is the crystallographic residual, R factor, which is defined as the summation of the difference between o b s e r v e d and calculated intensities divided by the summation of the o b s e r v e d intensities. The smaller this number, the better the structure is refined.

16

TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES

[1]

pG " ~C A)

C

°

G • G " A, •

U ° U •

D arm ~ O

•

, ~ .-'~\

/

c. A

AC a r m

G

AA arm

C~o U U

A A

T orm ~('~..o~ Gr,~A C A CI.~

"\

mC UsoG Ul-G1../n~

•

e U

~ I

Gso" mSC,o A

~m

•

V arm

~t'

A

®

Gm As A

A

[3

FIG. 6. (A) Nucleotide sequence of yeast phenylalanine tRNA arranged in a cloverleaf configuration. Bases that are conserved and semiconserved among most tRNAs are indicated by rectangles and circles, respectively. AA stands for amino acid, and AC for anticodon. Secondary base pairs (Watson-Crick type) are indicated by dots, and tertiary base pairs by solid lines. (B) Backbone structure of yeast phenylalanine tRNA. Shaded ribbons represent double helical stems.

T h e Structural F e a t u r e s of Yeast P h e n y l a l a n i n e T r a n s f e r R N A The structural features of yeast phenylalanine tRNA have been reviewed recently 3 and are summarized below. Overall Structure The crystal structure of yeast phenylalanine tRNA has an overall shape of the letter L, with the polynucleotide backbone of the molecule folded so that the acceptor stem and T stem form one continuous double helix with a gap, and the D stem and anticodon stem form another long double-helical arm with a gap. Each stem is an antiparallel, right-handed double helix similar to A-RNA. The relationship between the cloverleaf secondary structure and the three-dimensional backbone structure is shown in Fig. 6. The 3' end, where the peptide elongation occurs, is at one end of the molecule, and the anticodon, which recognizes the codon on the messenger RNA, is at the opposite end. The T loop, which has been implicated as a ribosomal R N A interaction site, appears at the corner of the L. These three functionally important sites therefore are 3 S.-H. Kim, in "Transfer RNA" (S. Altman, ed.). M.I.T. Press, Cambridge, Massachusetts, 1978. In press.

[1]

CRYSTALLOGRAPHIC DETERMINATION OF t R N A STRUCTURE

17

maximally separated, which may help minimize mutual interference between them and their corresponding sites on the ribosome.

Secondary Structure All four stems have a conformation similar to A - R N A , in that they have a shallow groove and a deep groove; the base pairs are considerably tilted from the helical axis; and the riboses adopt the C3'-endo conformation. The angle between the two helical axes of the acceptor stem and the T stem is 14°, and the corresponding angles between the D stem and the anticodon stem is 24 °. The angle between the two slightly bent long arms of the L is 92 °. All the base pairs in the stems are of the Watson-Crick type except for G(4).U(69).

Tertiary Structural Features There are many factors that contribute to the stability of the tertiary structure of tRNA, in addition to the base-paired stems described in the preceding section. There are hydrogen-bonding and stacking interactions between bases, hydrogen bonds between base and backbone and between backbone and backbone, and bonding between tRNA and essential metals and between spermine and water. Each of these is described and discussed separately below.

Base-Base Interaction. All the "tertiary" hydrogen bonding between bases has been recently reviewed. 3 None of the base-base tertiary hydrogen bonds is of the Watson-Crick type except the G19-C56 pair. All of these are located at the intersection of the two arms of the L. Nine tertiary base pairs are indicated in Fig. 6A, and their locations in the three-dimensional structure are shown in Fig. 7. Although only 55% of the bases in this molecule are in the doublehelical stem, the three-dimensional structure reveals that all except five bases (D16, D 17, G20, U47, and A76) are stacked. This dramatic stacking interaction is shown in Fig. 7B. Interaction Involving Backbone. Assignments of tertiary hydrogen bonds between bases are relatively easy because of the requirement for structural complementarity. However, assignment of hydrogen bonds involving the backbone is much more difficult. Based on the distance criteria and stereogeometry of hydrogen bonds, at least 25 hydrogen bonds can be assigned. 4 Many of these utilize the O2'-hydroxyl of riboses. 4 S. R. Holbrook, J. L. Sussman, R. W. Warrant, and S.-H. Kim, J. Mol. Biol. 123, 631 (1978).

18

TRANSFER RNA AND AM1NOACYL-tRNASYNTHETASES L

T arm 64

I

II

To~ 54

64

[1]

a(], qrrn - - I

~ 72

51 end

4o~

v Io;~ 1~/'~ 2o~ 38

anticodon

B

F]o. 7. Three-dimensional structure of yeast phenylalanine tRNA. a.a., amino acid; a.c., anticodon. (A) Ribose-phosphate backbone is shown as a long winding tube, and secondary base pairs as long bars. Tertiary base pairs are indicated by dark solid lines. (B) Backbone is shown as a thin wire. Long slabs represent secondary base pairs. Tertiary base pairs are indicated as bent slabs joined by a thick line. This figure shows the extent of base stacking. O t h e r L i g a n d I n t e r a c t i o n . Transfer R N A s are known to require sitespecifically bound magnesium ions to assume a functional conformation. We have located four sites where magnesium hydrates are tightly bound to the t R N A structure. Two of these sites are in the D loop, one is in the anticodon loop, and one is in the sharp bend formed by residues 8 through 12. These have been published p r e v i o u s l y ? From the locations of these magnesium hydrates, it can be assumed that their functional role is to stabilize the loops and sharp bends of the t R N A structure. Polyamines, such as spermine or spermidine, are known to stabilize the functional conformation of t R N A structure. We have used spermine in our crystallization and have located two possible candidate sites for spermine binding on t R N A . One is in the deep groove of the double helix formed by the acceptor and T stems, and the other is in the deep groove of the double helix formed by the D and anticodon stems. We have so far located approximately 60 water molecules bound to tRNA. Because of the weak electron densities for these water molecules, the reliability of the assignment is rather weak. The three-dimensional structure of t R N A therefore is stabilized by a

S. R. Holbrook, J. L. Sussman, R. W. Warrant, G. M. Church, and S.-H. Kim, Nucl. Acids Res, 4, 2811 (1977),

[1]

CRYSTALLOGRAPHIC DETERMINATION OF

tRNA

STRUCTURE

19

variety of interactions. Both arms of the L are primarily stabilized by the extensive base stacking and pairing of each double helix. At the center of the L where the two long arms meet, the structural stability and specificity are achieved not only by very extensive base stacking, but also by a very intricate hydrogen-bonding network between base and base, between base and backbone, and between backbone and backbone. In addition, three of the four magnesium ions are bound site specifically in this central region. The fourth magnesium ion is located in the anticodon loop, presumably stabilizing the conformation of that important region. 5,6 Functional Implications of the Three-Dimensional Structure The three-dimensional structure determined by the X-ray crystallographic method is necessarily a static picture of a molecule. Therefore, there is always concern whether the crystal structure is the same as the structure in solution. In the case of yeast tRNA Phe, almost all the results from solution studies are in good agreement with, and can be understood from, the crystal structure of the molecule. The solution studies involve varieties of techniques, such as small-angle X-ray diffraction, fluorescence energy transfer, complementary oligonucleotide binding, ultraviolet (UV)-induced photocross-linking, tritium labeling, base-specific chemical modification, and nuclear magnetic resonance (NMR). The sole exception comes from the laser light-scattering studies, where the diffusion coefficient of the molecule in a particular range of ionic strength appears to be very different from normal conformation. A more detailed description of the correlation between the crystal structure and the solution structure has been given recently.~'a

Molecular Design The overall shape of the tRNA molecule suggests some functional reason for it. The 3' end, where the amino acid or growing peptide is attached, is separated from the anticodon triplet by over 70 ,~, and these two functionally important sites are separated by more than 60/~ from a conserved sequence T-qJ-C, which has been implicated as a recognition site for ribosomal RNA (for a review, see Erdmann 9) located at the corner of the molecule. The three functionally important sites are therefore maximally separated within this architectural frame. Because these three sites may simultaneously interact with various ribosomal proteins 6 G. J. Quigley, M. M. Teeter, and A. Rich, Proc. Natl. Acad. Sci. U.S.A. 75, 64 (1978). r S.-H. Kim, Prog. Nucl. A c i d Res. Mol. Biol. 17, 181 (1976). 8 A. Rich and U. L. RajBhandary, A n n u . Rev. Biochem. 18, 45 (1976). a V. E r d m a n n , Prog. Nucl. Acid Res. Mol. Biol. 18, 45 (1976).

20

TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES

[1]

and RNAs, it probably is advantageous that they be separated to avoid mutual interference. Another aspect of the molecular design is the presence of a pseudo2-fold axis passing through the corner of the L at an angle, which relates one arm of the L to the other. The possibility that this symmetry is used in the general recognition or prealignment of aminoacyl-tRNA synthetase with tRNA has been noted earlier. 10

Structural Stability and Specificity Transfer RNAs are very stable and easily renaturable molecules. Such stability probably is essential to tRNA functionality. The three-dimensional structure of this tRNA reveals that 71 out of 76 bases are stacked, suggesting that base-stacking energy is a primary stabilizing factor. The specificity of the architectural framework, however, appears to be established by nine tertiary base pairs. Most of the bases involved in these tertiary base pairs are either conserved or semiconserved in all tRNAs, lending strong credence to the hypothesis that all tRNAs have the same basic framework. An additional factor that contributes to the stability of tRNA is the extensive involvement of the 2'-hydroxyl groups in forming a variety of hydrogen bonds. This may be a justification for the use of RNA instead of DNA for such structural nucleic acids as tRNA and ribosomal RNA.

Flexibility of the Molecule Using a least-squares procedures,ll we refined thermal parameters of each rigid group (ribose and phosphate) using X-ray diffraction data. To simplify the overall "thermal flexibility," the thermal parameters of the base, ribose, and phosphate of each nucleotide have been averaged to give one value per nucleotide, and these are shown in Fig. 8. The radius of each circle is proportional to the mean square displacement of each residue. The most prominent feature of this figure is the unusually high "thermal vibration" of the anticodon arm and the acceptor stem, suggesting that those two arms are more flexible than the other parts of the molecule. This molecular flexibility may have a functional rationale: When a tRNA participates in peptide elongation and translocation within the ribosome, it is likely that the acceptor stem and the anticodon arm move somewhat. l0 S.-H. Kim, Nature (London) 256, 679 (1975). 11 j. L. S u s s m a n , S. R. Holbrook, G. M. Church, and S.-H. Kim, Acta Crystallogr. Sect. A. 33, 800 (1977).

[2]

NMR FOR STUDYOF tRNA STRUCTURE

21

FIG. 8. E a c h nucleotide is r e p r e s e n t e d as a sphere. The radius of each circle is dra w n proportional to the " t h e r m a l v i b r a t i o n " of that residue.

Modified Nucleosides Examination of the modified bases shows that the modifications on the bases are not essential for maintaining the integrity of the tertiary structure of tRNA, suggesting that the modified bases probably are the recognition sites of various proteins that interact with tRNA. One of the roles of the modified base on the 3' side of the anticodon probably is to prevent any additional base pairing beyond codon-anticodon triplet pairs. There are two modified riboses, on residues 32 and 34, in this tRNA. Both are located in the anticodon loop, which is the most exposed loop of the molecule. The methylation of the 2'-hydroxyl of these sugars is probably to protect the most exposed region of the tRNA from random attack by ribonucleases, which require the 2'-hydroxyl group to form an intermediate for cleavage.

[2] T h e U s e o f N u c l e a r M a g n e t i c R e s o n a n c e i n t h e S t u d y o f T r a n s f e r R N A S t r u c t u r e in S o l u t i o n

By

BRIAN R. REID

High-resolution nuclear magnetic resonance (NMR) has found increasing application in the study of small nucleic acids, especially tRNA, over the last decade to the point where ;t is currently probably the most informative spectroscopic tool with which to probe the solution conformation and dynamics of these molecules. The fundamental principles underlying the phenomenon of magnetic resonance and its application to biochemical problems have been presented elsewhere in several excellent METHODS IN ENZYMOLOGY, VOL. LIX

Copyright© 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181959-0