Biochimie (199 I) 73. 887-897 © Soci6t6 fran~gaisede chimie et biologie mol6culaire / Elsevier, Paris

887

Review

Thermus thermophilus r i b o s o m e s for crystallographic studies MM Yusupov, MB Garber, VD Vasiliev, AS Spirin Institute of Protein Research, Academy of Sciences of the USSR, 142292 Pushchino, Moscow Region, USSR (Received 27 November 1990; accepted 14 March 1991)

Summary w Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, tile 30S ribosomal subunits, several ribosomal proteins, the elongation factor G and threonyl- and seryl-tRNA synthetases from a Gram-negative extreme thermophilic bacterium, Thermus thermophilus, .have been obtained at our institute. X-ray and neutronographic data from the 70S ribosome crystals have been collected up to 18 A and 60 A, respectively. Two-dimensional crystalline sheets of the 70S ribosomes have been studied by electron microscopy. Structural studies of crystals of 2 ribosomal proteins, LI and $6, elongation factor G and threonyi- and seryi-tRNA synthetases are also in progress. At present, Thermus thermophilus seems to be the most suitable microorganism to isolate ribosomes and their constituents for crystallographic studies. ribosome / ribosomal subunits I Thermus thermophUus I crystallization

Introduction

In 1980 HG Wittmann's group succeeded in the crystallization of ribosomal particles. They obtained 3dimensional crystals of the 50S ribosomal subunits from Bacillus stearothermophilus [1]. The first crystals were not perfect, but this result opened the field for structural investigations of the ribosome by crystallographic methods. Since then, significant progress has been made in obtaining crystals of ribosomal particles. X-ray quality crystals of the 50S ribosomal subunits [2-7], the 30S subunits [6-11] and, finally, the whole 70S ribosomes [9, 12] have been prepared. Structural investigations of ribosomes and ribosomal subunits are underway [6, 13]. These achievements are based, at least in part, on the use of thermophilic and halophilic bacteria as sources of ribosomes. Bacillus stearothermophilus and Halobacterium marismortui have been found to be good sources for the crystalline 50S ribosomal subunits; Thermus thermophilus is best for the crystalline 70S ribosomes and 30S ribosomal subunits. A long-term program on the purification and crystallization of ribosomes and other components of the protein-synthesizing system from T thermophilus (Gram-negative extreme thermophilic eubacterium with an optimal growth temperature of + 75°C) was set up at the Institute of Protein Research (USSR) about 10 years ago [14]. Proteins and nucleic acids

from this organism are very stable and can be crystallized more easily than those from mesophilic and moderate thermophilic organisms. Using this bacterium, we managed to obtain good 3-dimensional crystals of the 30S ribosomal subunits and the 70S ribosomes [8, 9, 11-13]. Within the framework of this program, besides crystals of ribosomal particles, we also obtained X-ray quality crystals of elongation factor G, threonyl- and seryl-tRNA synthetases, and ribosomal proteins L I and $6. Structural investigations for all of these are in progress [ ! 5-19]. Within the framework of the program, sequences of T thermophilus 16S RNA [20], elongation factor G and 2 ribosomal proteins [21, 22] have also been determined at this institute. Characteristics of T thermophilus ribosomes

Purification of ribosomes and ribosomal subunits Ribosomes for structural studies must meet the following standards. First, they must be free from contamination by proteins, nucleic acids and other cell components. Second, ribosomes must be structurally homogeneous. Third, the structure must be native. According to the technique developed for E coli ribosomes, the purification of the particles from foreign macromolecules and non-specifically adsorbed cellular components can be best achieved by

888

MM Yusupov et al

centrifugation at a high salt concentration or by chromatography [23-25]. The pure ribosomes can then be subjected to fractionation. For instance, the ribosome fraction that does not dissociate into subunits at 6 mM Mg2+ can be isolated (tight couples [26]); the ribosome samples prepared in such a way are more or less structurally homogeneous and seem to be 'native'. The method of purification for T thermophilus ribosomes differs from standard methods for purification of E coil ribosomes. Removal of a yellow membrane fraction is the most difficult step. Ribosomes washed only by centrifugation at a high salt concentration [27, 281 contained a yellow p~gment and additional proteins. Our procedure of ribosome purification from membrane contaminations and other cell components is based on centrifugation of ribosomes through a high density CsCI-sucrose cushion [29]. The ribosomes twice washed in this manner had no additional proteins and nucleic acids. For further purification and isolation of homogeneous ribosomes, methods of centrifugation in a sucrose gradient or chromatography and differential Mg2+-depletion dissociation were also used. Dissociation of T thermophilus ribosomes upon decrease of magnesium ion concentration was found to depend strongly on the purity of the ribosomal samples. Figure 1 demonstrates the dependence of dissociation of ribosomes prepared by different methods [28, 29] on the concentration of magnesium in solution. The curve of E coli tight couple dissociation [30] is given for comparison. It is seen that half-dissociation of crude ribosomes prepared without washing in high salt concentration takes place at 3 mM Mg2., while that of washed ribosomes is at 12.5 mM Mg2.. This considerable difference in ribo-

oE I00 e'~

•:- 50 03

o I

I

I

I

I

I

I

2

3

5

I0

20

50

50

mM MqCl z

Fig 1. Dependence of T thermophilus ribosome dissociation on the concentration of magnesium ions. o, ribosomes washed through CsCl-sucrose cushion; e, tight couples prepared from ribosomes washed through CsCl-sucrose cushion; A, crude ribosomes; &, control tight couples from E coli.

somal samples can be tentatively explained by the presence of a stabilizing factor in crude ribosomes; this factor can be lost during ribosome purification. The similar slight slopes of the dissociation curves for crude and washed ribosomes indicate the heterogeneity of the samples. Homogeneous samples of ribosomes can be prepared by selection of tight couples from washed ribosomes. The dissociation curves of these ribosomes and E coli tight couples have the same steep slope [31 ]. T thermophilus ribosomal subunits from ribosomes washed through CsCl-sucrose cushion were isolated by sucrose gradient centrifugation in a buffer with 10 mM MgCI2 and 400 mM NaCI, or, in the case of tight couples, in a buffer with 1 mM MgCI2 and 100 mM NH4CI. Structural and functional differences between these isolated subunits were not detected. Furthermore, it was shown that the sedimentation coefficient of 30S subunits did not change even in a solution without magnesium ions, though the sedimentation coefficient of 50S subunits in this case decreased to 48--46 S. The electron microscopic images and sedimentation coefficients of ribosomes and ribosomal subunits from T thermophilus and E coil were shown to be the same. Cell-free translation system from T thermophilus The poly(U)-dependent cell-free system of polyphenylalanine synthesis was first reported by Onolwashita et al [27]. They used ribosomes twicewashed with 1 M NH4CI, a total enzyme fraction (S-100) freed from nucleic acids, and Phe-tRNA preliminarily acylated by [~4C]phenylalanine in the presence of the S-100 fraction. Some difference of this system was shown in comparison with the ceilfree translation system of E coll. The functional activity of T thermophilus ribosomes depended on the presence of magnesium ions and spermine. The optimal concentration of magnesium ions was 10 mM in the presence of 3 mM spermine. Without spermine the functional activity of ribosomes was lower, and it was necessary to increase the magnesium ion concentration to 20 mM and to pre-incubate the mixture of ribosomes, poly(U) and Phe-tRNA at 10°C. Later the cell-free translation system from T thermophilus was optimized by adjusting the concentration of all the components [29]. The ribosomes used in the optimized system were purified through the CsCl-sucrose cushion. Cold preincubation was required for this system even in the presence of spermine. The yield of polyphenylalanine synthesis in the poly(U)-dependent cell-free translation system of T thermophilus was ~ 6 pmol of incorporated phenylalanine per 1 pg ribosomes and did not differ from the synthesis yield of the E coli system (fig 2) (Shirokov and Kolb, personal communication).

Thermus thermophilus ribosomes for crystallographic studies

° l

"E

6.0

g.

4.5

~

3.0

>,

1.5

2 5 I0

20

40

min

Fig 2. Polyphenylalanine synthesis in the poly(U)-depend-

ent cell-free translation system, o, T thermophilus at 65°C; e, E coil at 37°C.

Ribosomal RNAs Sequences of 5S RNA [32], 16S RNA [20] and 23S RNA [33] were determined, and a high homology of the sequences of ribosomal RNAs from T thermophilus and E coli was shown. For example, the homology of 16S RNA is 75%, and a computer model of its secondary structure (fig 3) does not differ from the model of the E coli 16S RNA [34]. The differences in the sequence are observed mostly in the secondary structure hairpins where some AU pairs are changed for the GC pairs. Substitutions, deletions and insertions are seen predominantly in variable parts of the structure. In the conservative parts, presumably participating in binding of ribosomal iigands (antibiotics, tRNAs, mRNAs and others), there are virtually no differences. 5S RNA from T thermophilus was crystallized [35], but unfortunately the crystals were not good enough for structural studies.

Ribosomal proteins Analysis of ribosomal proteins was carded out by 2-dimensional electrophoresis [29] (the ribosomes used were washed through CsCl-sucrose cushion). Figure 4 shows the results of electrophoretic analysis of ribosomal proteins of 70S ribosomes, 50S and 30S ribosomal subunits from T thermophilus and 70S ribosomes from E coli. The total ribosomal protein of the

889

large subunit was divided in this system into 30 spots, and that of the small subunit into 20 components. Ribosomal proteins are numbered on the electropl.oregrams, as shown in figure 4. The analog of ribosomal protein S 1 of E coli is absent from the 30S subunit of T thermophilus, so that the protein numeration of the small subunit begins from protein TS2. In the total ribosomal protein of 70S ribosomes proteins X l and X2 were detected, but they were abse.~t among proteins of the isolated 30S and 50S ribosomal subunits. Isolation of ribosomal subunits by centrifugation in the sucrose gradient seems to result in the loss of these proteins. The electrophoretic mobilities of ribosomal proteins from T thermophilus and E coli are similar, indicating small differences of the isoelectric points and molecular masses of corresponding proteins. Two-dimensional electrophoresis only, however, does not permit precise identification of the T thermophilus proteins corresponding to those of E coli. For many proteins, identification can be performed from N-terminal sequences, molecular masses, and their locations on the electrophoregram. In this manner, 6 T thermophilus ribosomal proteins have been identified: L1, L6, L9, $5, $6 and S12 (SE Sedelnikova, unpublished observations). There are no difficulties in identifying the pentamerit complex of proteins (L7/L 12)4L 10 which forms the stalk in the 50S ribosomal subunit. When the complex is selectively extracted the stalk disappears. It was shown by electron microscopy with T thermophilus ribosomes (Agalarov and Ryazantsev, unpublished observations). The pentameric complex contains 2 of the most acidic proteins of the 50S subunit and is characterized by the molecular ratio of the 2 polypeptide chains of 4 to !. SDS electrophoresis has demonstrated, however, that the molecular masses of each polypeptide in the T thermophilus complex are somewhat higher than those in the E coli complex [14]. Equilibrium sedimentation data have also demonstrated that the molecular mass of the whole pentameric complex from T thermophilus is 20% higher than that in E coli (SE Sedelnikova, unpublished observations). Special procedures of selective removal and purification of ribosomal proteins from T thermophilus have been developed [36, 37]. More than half of the individual ribosomal proteins have been purified in a preparative scale, and some of them have been crystallized [37, 38]. Structural investigations of ribosomal proteins are to be carded out in parallel with crystallographic studies of ribosomal particles to provide more detailed information on ribosome structure. At present, X-ray analysis is already underway for the proteins L I and $6 from T thermophilus (figs 5, 6) [ 18, 191.

e,.I

I | I ol

9

,,.,]

:>

2:

o

c:r"

:2.

0'~

0

I=

0

("I)

,=.:0r~

•"~ ,.ej

-

w.

|11

C

- G'~

~_ ~, ~")-n

I II

I

I

6") ~ 0 6 " I , r l

lel

|lie|

)n

.,.'b_

--

°

w

"~

O--t;')"

- - " - JE- ~ ' ' ' ' -

6")._

R'~ C ([;

o~

-~,.)

%

1,,

~

~.

~tl"....~"--P_e--(;~

~.., ~.,v.., ....

,~-~

--

~

~'-~".._n.~

--';..'_~ C

ca~)

'.;"

G') - t ' ) b'~-t'l

~'~

('1 - k') 6"1 - ('~ G'~ e C : t ' l - b~l

_~,~c~.,~c,,~,',n~c~'~'~

~]~....

le

J)r'~ I ~ C

c

.

~--~'..'1~ ~

"~,,~

-. _~ c,~-~_ -,",~' ~ ~

,.,,. . . . .

""

. , ~ , c - =,,.,,

n_~

n-~

•.

I | I I I •

_"-'~L

,~.~

--~-~

c c

.'%



_-,~

~

~i

........

• • . . , ,

c

~ nc,~":

c

c

t'~

•'

4"p

-

1~

~

~

~

P

c

o_

...]1,1.

""

C

!1'

I I

~:

0 ~

~m,~"

_nqL';/':'~c ~n

c

~-CI

~" ~

¢,

~ n~c~r'~ iii i II



c

I

____

o _J,,]l,,_~.......-

o-%

-&.~

~ I~

"~..c,

]Dr-

o

n~.c

be,-

~=/"/""--

.~,,,"P'. C,

I

ii

-

|ii

o,.,~

:,-c

-~-~

....

~:~ "%

C'J-- ~'~

6")

]~

c',~

-~.,

c-~,

n-c~

6"1

('I--

_~_'"--

~ .

C

c

o

~"1~--

o

u u u , , "

n(',j~G'~ of (,~

~=,,r~nc~c~c"=,~r.,, ii lllol llell



~

~

]p,

~..,~,,,

I I I I | I I I • •"

-,r

"~'""

~

-,.

--~_

,n -,--o co~ ~_

C,~P

~-n

~

~;'c

~

-c£..:'~

c

_0

COq;~

'~'.-cL. L

w~'t/o ~

.,.JD, - c,._

~,-c

%__

~

_,,"':''-'@ I I

-~,,~c~c~.,~c iiii Olll

~ G~

~c

6") (-~ •

.J~--C;; ~

_~-.:%.,

6"1 -

n-~

I eel

n

, ,~_

v.~

_~

..~

~ ,,.

¢-,~o-,nq.,., _ c, ~_-~' -~'--_~ ~o _ _ ~ ' d~' ~ - ~ "

~;~c~

"~_ - ~



n

t.,.

~ %_~"~

nC('14~k'ICk~

n'L la,~" " b ' )

~.~

P-t")

-

-c,.

q~,,_

"-

"'~

~ ]~"

; ,_"

.~:.',,F

~'

""

"'~""

,~.r~'.",-, c,"' ".-_

~.,)// ,-.

~



~,-~ . ~ _.. C J k'tr-~,,

~'..~J;'-

~, _~',,..

~,-

~wm'~('ll

; , : _ ~ ,.~.-:,~v'

k'l-nc;

~"~b'~t~

s-t"L

~

-

[*~

~-',,--,,~

"

"

~

~..

O/o

..c .... ...~" ~

• '-

c

~

~,

q~l

..'~

~

cc-~_

cn-o

~-

~ , ~

~. ' ~ , ~ = ~ - . ~ ~

,,,,,

""

~m,*~cx:

_~

t,~ ~,

Cl]1~

..

~:,~

m

¢'I

~,

0

0

Thermus thermophilus ribosomes for crystallographic studies.

Three-dimensional crystals of the 70S ribosomes, the 70S ribosome-mRNA-tRNA complex, the 30S ribosomal subunits, several ribosomal proteins, the elong...
3MB Sizes 0 Downloads 0 Views