Molec. gen. Genet. 144, 273-280 (1976) @ by Springer-Verlag 1976
Surface Topography of the Bacillus stearothermophilus Ribosome Hugh M. Miller and S. Marvin Friedman Department of Biological Sciences, Hunter College of the City University of New York, New York, N.Y., U.S.A.
David J. Litman and Charles R. Cantor Departments of Chemistry and Biological Sciences, Columbia University, New York, N.Y., U.S,A.
Summary. The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62-78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were $4, $7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins $2, $3, $7, S13, Sa, L5/9, L10, Lll and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including $5, $9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.
Introduction
Ribosomes from the thermophilic bacterium, Bacillus stearothermophilus, are of particular interest because they differ from Escherichia coli ribosomes in several important properties, including heat stability (Friedman et al., 1967) and the translation of polycistronic messenger RNA's (Lodish, 1969). Both protein S12 and 16S RNA have recently been implicated in the failure of thermophile ribosomes to translate the coat protein and replicase cistrons of bacteriophage R17
RNA (Held et al., 1974). Although the molecular basis for the unusual heat stability of thermophile ribosomes remains unknown, reconstitution studies with mixtures of E. coli and thermophile ribosomal components revealed that B. stearothermophilus ribosomal proteins play a key role in protecting the ribosome from thermal denaturation (Nomura et al., 1968). Experiments employing reconstruction, immunological, chemical and genetic approaches have established correspondence between many E. coli and B. stearothermophilus ribosomal proteins (Horne and Erdmann, 1972; Fahnenstock et al., 1973; Geisser et al., 1973; Isono et al., 1973; Tischendorf et al., 1973; Higo and Loertscher, 1974; Isono, 1974; Visentin et al., 1974; Yaguchi et al., 1974). Since lactoperoxidase-catalyzed iodination has proven to be a valuable technique for probing the topography of E. coli ribosomal proteins (Michalski et al., 1973; Miller and Sypherd, 1973; Litman and Cantor, 1974; Litman et al., 1974; Michalski and Sells, 1974, 1975), it was of interest to apply this method also to thermophile ribosomes. One would then be in a position to ask if tyrosines in homologous ribosomal proteins from these different bacterial species occupy similar sites within the ribosome. In addition, information concerning the structural organization of thermophile ribosomal proteins could lead to a clearer understanding of ribosome stability. In the present report, we have utilized enzymatic iodination to study the surface topography of the B. stearothermophilus 70S ribosome and isolated subunits. The results are interpreted in regard to proteins with predominantly buried and exposed tyrosine residues, proteins involved in the 30S:50S interface region and conformational changes in proteins attendant on subunit interaction. A comparison of these classifications for some homologous ribosomal proteins from B. stearothermophilus and E. coli is presented.
274
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
Materials and Methods Buffers Buffers of the following composition were used in this study: TMAI (0.01 M Tris-HC1, pH 7.5, 0.01 M Mg acetate, 0.03 M NH4C1, 0.006 M 2-mercaptoethanol); TMA-II (0.01 M Tris-HC1, pH 7.5, 0.0003 M Mg acetate, 0.03 M NH4C1, 0.006 M 2-mercaptoethanol); TMN-II (0.01 M Tris-HC1, pH 7.5, 0.001 M Mg acetate, 0.1 M NH4C1, 0.006 M 2-mercaptoethanol).
Preparation of Ribosomes B. stearothermophilus strain 2184 was grown at 65 ° C to midlogarithmic phase and the S-30 fraction was prepared as previously described (Friedman and Weinstein, 1966). All of the following procedures were carried out at 4 ° C. The S-30 fraction was layered onto a buffered 1.1 M sucrose-0.5 M NH4C1 solution (Fahnestock and Nomura, 1972) in the ratio of 1 volume S-30 to 3 volumes sucrose. Ribosomal particles were pelleted by centrifugation at 28,000 rpm for 16 h in a Spinco 42.1 rotor and resuspended in TMA-I buffer. 70S ribosomes were purified by centrifugation on 7-30% linear sucrose gradients in TMA-I buffer at 20,000 rpm for 16 h in a Spinco SW 25.1 rotor. Fractions (1 ml) were collected by withdrawing the contents from the bottom of each tube and only those fractions containing 70S ribosomes free of 50S subunits were pooled. Pure 70S ribosomes were pelleted and resuspended in TMA-I buffer at a concentration of approximately 20 mg/ml. 70S ribosomes were dissociated by dialysis against TMN-II buffer for 40 h and the subunits were separated on 10-25% discontinuous sucrose gradients centrifuged in a Spinco SW 25.1 rotor at 22,000 rpm for 16 h. Pure subunits were obtained by aspirating the 30S subunit band located near the middle of the tube and fractionating the more diffuse 50S subunit band located toward the bottom of the tube. When small amounts of subunits were to be separated from iodinated 70S ribosomes, a 7-25% linear sucrose gradient was centrifuged in a Spinco SW 27 rotor at 20,000 rpm for 17 h. Analytical gradients (5-20% sucrose) were centrifuged in a Spinco SW 50.1 rotor at 45,000 rpm for 90 min.
Iodination of Ribosomes 70S, 30S and 50S particles were dialyzed against TMA-I buffer minus 2-mercaptoethanol and iodinated according to the procedure of Litman and Cantor (1974). The specific activity of the Na 125I used was 500 mCi/mM (New England Nuclear). Typical reactions contained t0 mg of 70S ribosomes, 4 mg of 30S subunits or 8 mg of 50S subunits. After the reaction was stopped by the addition of 2-mercaptoethanol, the particles were pelleted, resuspended and dialyzed against TMA-II buffer. Iodinated 70S ribosomes were separated into 30S and 50S subunits as described above. Unlabelled subunits were added to these derived subunits and to iodinated free 30S and 50S subunits to yield final concentrations of 17 mg 30S subunits and 28 mg 50S subunits.
Extraction of Ribosomal Proteins 30S and 50S subunits were stripped of protein by the acetic acid method of Hardy et al. (1969).
Two-dimensional Gel Electrophoresis and Sample Counting Two-dimensional gel electrophoresis of ribosomal proteins was carried out according to Kaltschmidt and Wittmann (1970) except
that the first dimension gel was immersed in 0.3 M HC1 for 5 min in order to effect the pH change (Avital and Elson, 1974). After staining the gel slabs in 1% Amido Black and 7% acetic acid and destaining with 7% acetic acid, the protein spots were carefully cut out with a scalpel and placed in scintillation vials. The samples were treated and counted as previously described (Litman and Cantor, 1974).
Protein Synthesis Assay The activity of ribosomes and subunits was assayed by poly Udirected phenylalanine incorporation (Nirenberg, t963) under conditions of limiting ribosomes as described by Litman and Cantor (1974).
Results
Iodination of Ribosomal Particles 70S, 30S and 50S ribosomal particles were iodinated to less than saturation levels in parallel reaction mixtures according to the procedure of Litman and Cantor (1974). The iodinated 70S ribosomes were then dissociated and pure 30S and 50S subunits were isolated by sucrose gradient sedimentation. The specific activities of these subunits were compared to those of subunits iodinated in the free state. Iodinated free 30S subunits and 30S subunits derived from iodinated 70S ribosomes had almost identical iodine contents of 22.1 and 22.0 iodines per particle, respectively. The free 50S subunit incorporated 29.6 iodines per particle, whereas the 50S subunit derived from iodinated 70S ribosomes incorporated 25.1 iodines per particle. Thus, the free 50S subunit incorporated about 18% more 125I than did the 50S moiety of the 70S ribosome. Similar labeling data were reported by Litman and Cantor (1974) for the E. coli 50S subunit.
Effect of lodination on Ribosome Activity and Sedimentation Properties In order to obtain meaningful structural information for ribosomes from experiments employing a topographical probe, the chemical modification should not significantly alter the functional activity of the particle nor cause it to undergo extensive conformational changes. Therefore, the protein synthesis activities of iodinated and unlabeled 70S, 30S and 50S particles were compared. Ribosomes and subunits were iodinated to the same extent as those which were used for two-dimensional gel electrophoresis of ribosomal proteins except that low specific activity (1 mCi/mM) 12sI was used. The intrinsic trichloroacetic acid precipitable counts, which never amounted
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
to more than 5% of the total counts, were substracted from the counts obtained in the protein synthesis assay. Table 1 shows that 70S particles retained 79% of the control protein synthesis activity after they had been heavily labeled (40 iodines/particle). 30S subunits labeled to the extent of 22 iodines/particle retained 62% of the control activity and 50S subunits labeled to the extent of 29 iodines/particle retained 72% of the control activity. Figures 1A and B show sedimentation profiles of iodinated and unlabeled 30S and 50S subunits. The sedimentation velocities were found to be virtually identical for both iodinated and unlabeled particles, indicating that no gross conformational distortion had occurred as a result of the covalent reaction.
Approximately two milligrams of ribosomal proteins were used for each two-dimensional gel separation. Figures 2A and B show typical two-dimensional gel electrophoretic patterns of 30S and 50S subunit proteins from B. stearothermophilus strain 2184. The 30S pattern of 22 isolated proteins was virtually identical to that obtained by Isono and Isono (1975) for B. stearothermophilus strain 799 and their numbering system was employed to identify the spots. In the case of 50S pattern, most of the 44 proteins corresponded to the 38 proteins found in strain 799 by Horne and Erdmann (1973) and their numbering system was employed to identify the matching spots. The six extra proteins present in strain 2184 but absent in strain 799 were numbered LX1 through LX6. Spots corresponding to proteins S1 and LX4 were often too faint to observe and could not always be cut out of gels. These proteins may be only weakly associated with ribosomes and thus partially removed during the isolation procedure as has been shown
Table 1. Effect of iodination on the protein synthesis activity of B. stearothermophilus ribosomal particles
70S 70S 30S 30S 50S 50S
Iodines/ particle
0 47 0 22 0 29
!.6
4.0
A260
0.8
_.jl 4
f
cpmxlO 3 2.0
8 12 16 fraction number
20
S 2.5
2.0
cpm x 103,
A26o
Identification of Iodinated Ribosomal Proteins
Particle
275
Activity a Poly U-phe incorp. (cpm x 105/mg particle)
Per cent
3.9 3.1 7.8 4.8 5.6 4.1
100.0 78.8 100.0 62.0 100.0 72.0
a Assay conditions for poly U-directed phenylalanine incorporation are referred to in Materials and Methods.
1.2
1.5
0.4
0.5 J t
4
8
I
12 fraction
16
number
20
Fig. 1 A and B. Sucrose gradient sedimentation profiles of untreated (triangles) and iodinated (circles) (A) 30S and (B) 50S subunits from B. stearothermophilus
to be the case for E. coli protein S1 (Nomura, 1973). Several protein spots were not sufficiently resolved to be cut from the gels as individual proteins and were therefore excised as pairs or triads. Proteins treated in this manner were S15/16, L5/9, L7/8, L12/ 14/15, L16/19/20, L24/25, L30/31, L34/35 and L37/38. Table 2 shows the radioactivity data obtained for proteins that were labeled in isolated 30S subunits and in 30S subunits derived from labeled 70S ribosomes. Table 3 shows the radioactivity data obtained for proteins labeled in isolated 50S subunits and in 50S subunits derived from labeled 70S ribosomes. The values presented are the averages from six individual gels in each case. The radioactivity of each protein has been corrected for the total amount of protein that actually ran on to the gel as well as for the specific activity of each subunit according to the calculations described by Litman and Cantor (1974). The radioactivity data presented in Tables 2 and 3 has been processed two ways. One calculation expressed the difference between the extent of iodination (expressed as tyrosine equivalents) of each protein in isolated subunits and the extent of iodination of each protein in subunits derived from iodinated 70S ribosomes. A second calculation expresses the
276
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome Fig. 2A and B. Two-dimensional electrophoretic patterns of B. stearothermophilus strain 2184 (A) 30S and (B) 50S ribosomal proteins
ratio of the extent of iodination (expressed as tyrosine equivalents) of each protein in isolated subunits to the extent of iodination of each protein in subunits derived from iodinated 70S ribosomes. Only proteins which have been substatially iodinated (> 800 cpm) have been treated in this manner in order to eliminate values which may not be statistically significant. It can be seen from Tables 2 and 3 that the extent of iodination varies considerably for individual proteins in each subunit. 30S proteins that have been extensively labeled include $4, $7, S10, S15/16 and Sa, whereas 50S proteins that have been extensively labeled include L2, L4, L5/9, L36. These proteins presumably have several tyrosines exposed on the surface of the ribosome and are thus readily accessible
to lactoperoxidase-catalyzed iodination. On the other hand, proteins that have been labeled to a small extent in 30S and 50S subunits include S14, $20, L3, L7/8, L12/14/15, L13, L27, L37/38, LXs and LX 6. These proteins must be unreactive to enzymatic iodination either because their tyrosine content is low or their tyrosines are buried deep within the ribosome structure. Heavily iodinated proteins that have been labeled more in isolated 30S subunits than in 30S subunits derived from 70S ribosomes are $3, $4, $7, S13 and Sa, whereas those that have been labeled more in isolated 50S subunits than in 50S subunits derived from 70S ribosomes are L2, L5/9, L10, L l l , L24/25 and LX1. Since these proteins are less reactive to
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
enzymatic iodination in the intact 70S ribosome than in the free subunits, they may be associated with the 30S:50S interface region. Heavily iodinated proteins that have been labeled more in subunits derived from 70S ribosomes than in isolated subunits are $5, $9, S12, S15/16, S18 and L36. These results indicate that conformational changes occur in or around these proteins when subunits associate to form the 70S ribosome.
Discussion
Lactoperoxidase-catalyzed iodination is generally considered to be a probe of surface topography because the large size of the enzyme (M.W. 78,000) would preclude its ability to penetrate the ribosome structure. Support for this contention comes from the iodination data reported by Litman and Cantor (1974) for the E. coli 50S ribosomal proteins. They, demonstrated that heavily labeled proteins did not constitute a specially selected class of tyrosine-rich proteins. It should be noted that in the case of the present results for B. stearothermophilus, there also does not appear to be any correlation between the extent of iodination and the tyrosine content of the
277
ribosomal proteins. Since the quantitative amino acid composition of isolated ribosomal proteins from B. stearothet~nophilus strain 2184 has not yet been determined, such a comparison must be based upon the quantitative amino acid data reported by Isono and Isono (1974) for the 30S ribosomal proteins from B. stearothermophilus strain 799. Their analysis showed, for example, that B-S19 had a high tyrosine content, whereas no tyrosine was detected in B-S10. In the present study, we found that B-S19 was labeled to a very small degree, whereas B-S10 was labeled to a relatively large degree. The extensive labeling of B-S10, presumably a tyrosine-free protein, was at first difficult to interpret. However, a careful examination of the sequence data (Yaguchi et al., 1974) for the first 15 amino acids starting at the N-terminal position of B-S10 derived from B. stearothermophih~s strain 10 revealed the presence of two tyrosine residues. It remains to be seen whether these variations are due to differences in the analytical techniques employed or in the amino acid composition of homologous proteins from different strains of B. stearothermophilus. The absolute counts found in a protein spot will be limited by the number of tyrosine residues present in that particular protein. This can best be illustrated
Table 2. 1251 Labelling of B. stearothermophilus 30S ribosomal proteins Protein
B-S1 B-S2 B-S3 B-S4 B-S5 B-S6 B-S7 B-S8 B-S9 B-S10 B-S11 B-S12 B-S13 B-S14 B-S15/16 B-S17 B-S 18 B-S19 B-S20 B-S21 B-Sa
cpm 30S ( x 10 -2)
11.8 12.8 46.2 70.9 35.2 39.7 343.1 40.5 20.0 81.4 8.2 36.8 21.6 1.4 81.5 6.8 45.9 9.3 1.2 4.9 74.3
Tyr. Equiv.
0.26 0.28 1.03 1.58 0.78 0.88 7.62 0.90 0.44 1.81 0.18 0.82 0.48 0.03 1.81 0.15 1.02 0.21 0.03 0.10 1.65
cpm 30S via 70S ~ ( x 10 -2) 12.8 5.4 36.2 61.4 58.9 34.6 278.2 45.6 33.8 77.9 10.6 51.0 9.2 1.9 129.6 15.2 63.8 9.4 1.4 4.0 60.7
Tyr. Equiv.
0.28 0.12 0.80 1.36 1.31 0.77 6.18 1.01 0.75 1.73 0.24 1.13 0.20 0.04 2.88 0.38 1.42 0.21 0.03 0.09 1.35
ATyr. b
30S 30S via 70S
-0.16 0.23 0.22 -0.53 0.11 1.44 -0.11 -0.31 ---0.31 0.28 . . -1.07 -0.23 - 0.40 -. . . . 0.30
2.33 1.29 0.60 1.23 -0.59 0.75 0.73 2.40 .
.
. 0.63 0.39 0.72 --
. .
. .
. . 1.22
Iodines/30S=22.1 ; Iodines/30S via 70S=22.0. b c
30S subunits isolated from iodinated 70S ribosomes. (30S--30S via 70S) tyrosine values less than 0.1 are denoted by - - . Tyrosine ratios within the range of 0 . 8 - 1.2 are denoted by - - ; ratios for proteins with low levels of labeling are denoted by .... .
H . M . Miller et al: Topography of the
278
Bacillus stearothermophilus Ribosome
Table 3. 1251 Labelling of B. stearothermophilus 50S ribosomal proteins Protein
cpm 50S ( x 10- z)
Tyr.
c p m 50S
Equiv.
via 70S a
Tyr. Equiv.
ATyr. b
50S 50S via 70S
0.38 1.74 0.05 1.40 9.15 1.20 0.04 0.37 0.53 0.03 0.01 2.22 0.55 0.28 0.09 0.10 0.59 0.31 0.19 0.04 0.65 0.11 0.08 0.06 0.03 0.33 2.33 0.04 0.36 0.23 0.19 0.05 0.04
0,13 0.21 . . 0.14 3.35 0,19 . . 0.29 0.27 . . . . ---. . . . -0.43 0.10 . . 0.17 ----
1.34 --
( x 10 -2) B-L1 B-L2 B-L3 B-L4 B-L5/9 B-L6 B-L7/8 B-L10 B-Lll B-L12/14/15 B-L13 B-L16/19/20 B-L17 B-L18 B-L21 B-L22 B-L23 B-L24/25 B-L26 B-L27 B-L28 B-L29 B-L30/31 B-L32 B-L33 B-L34/35 B-L36 B-L37/38 B-LX 1 B-LX2 B-LX 3 B-LX5 B-LX 6
21.4 82.1 3.5 64.9 528.4 58.7 2.8 27.7 33.8 2.0 1.0 89.7 26.5 13.2 5.1 6.3 27.4 31.0 12.3 1.7 34.6 5.0 5.0 4.0 2.3 14.9 81.9 1.7 24.3 13.9 14.4 2.8 2.3
0,51 1,95 0.08 1.54 12.50 1.39 0.07 0.66 0.80 0.05 0.02 2.13 0.63 0.3• 0.12 0.15 0.65 0.74 0.29 0.04 0.82 0.12 0.12 0.10 0.05 0.35 1.94 0.04 0.58 0.33 0.34 0.07 0.06
16.2 73.3 2.2 59.2 385.9 50.4 1.5 15.4 22.4 1.1 0.5 93.8 23.2 11.9 3.8 4.4 24.7 13.2 7.9 1.7 27.6 4.5 3.3 2.5 1.2 13.8 98.1 1.7 15.0 9.6 8.0 0.5 0.4
. . 0.39 . . 0.22 0.10 0.15 . . . .
.
.
. -1.37 --
.
.
. 1.78 1.51
. .
. .
. . ----
. .
. .
. . -2.39 1.53
.
. 1.26 - --....
.
.
.
.
.
.
0.83 1.61 1.43 1.79 . .
. .
. .
I o d i n e s / 5 0 S = 2 9 . 6 ; Iodines/50S via 7 0 S = 2 5 . 1 , a b c
50S subunits isolated from iodinated 70S ribosomes. ( 5 0 S - 50S via 70S) tyrosine values less than 0.1 are denoted by - - .
Tyrosine ratios within the range of 0.8-1.2 are denoted by - - ;
ratios for proteins with low levels of labeling are denoted by .... .
in the extreme case of proteins which have been shown to be devoid of tyrosine, namely E-L7 and E-L12 (Mora et al., 1971) and B-L7 and B-L12 (Visentin et al., 1974). Litman and Cantor (1974) found that very few counts were incorporated into E-L7 and E-L12, and very low labeling levels were also observed for B-L7 and B-L12 in the present study. The topography of these important ribosomal proteins cannot be revealed by enzymatic iodination and will have to be determined by other techniques. The results obtained from the present experiments provide evidence for the structural organization of proteins within the B. stearothermophilus ribosome. Heavily labeled proteins, notably $4, $7, S10, Sa, L2, L5/9, L6 and L36, are likely to have a large fraction of their tyrosine residues exposed on the surface of the ribosome. Proteins that were labeled more
extensively in isolated subunits than in the intact 70S ribosome include $2, $3, $7, S13, Sa, L5/9, L10, Lll and L24/25. These proteins are either associated with the 30S:50S interface region or are affected by conformational changes when subunits interact resulting in the masking of some tyrosine residues. Proteins that were more heavily labeled in the intact 70S ribosome than in the isolated subunits include $5, $9, S12, S 15/16, S 18 and L36. This suggests that these proteins undergo or are affected by conformational changes when subunits combine resulting in enhanced exposure of some tyrosine residues. Such a relaxation or expansion of ribosomal structure is particularly evident in the case of the 30S subunit. A similar effect has also been observed for the E. coli 30S subunit by Litman et al. (1974). The availability of the data detailed above now
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome Table 4.Topography of B. stearothermophilus and E. coli ribosomal proteins as determined by enzymatic iodination Location
B. stearothermophilus "
E. coli b
Highly exposed
$4, $7, S10, Sa
$5(6) c, $7(7), $9(10), S18(19)
L2, L4, L5/9, L6, L36
L2(2) d, L5, L6(5) e, L10(10) d, L11, L26
30S:50S Interface
$2, $3, $7, S13, Sa
$10(13)
L5/9, L10, L l l , L24/25
L2, L13(13) a, L18(22) ~, L26, L27, L28
Conformational changes upon 70S formation
$5, $9, S12, S15]16, S18
$3, $6(9), $9, S 18(19)
L36
Lll
" Data from the present study. b Data of Litman et al. (1974) for the 30S proteins and of Litman and Cantor (1974) for the 50S proteins. c Figures in parentheses indicate homologous B. stearothermophilus 30S proteins according to Yaguchi et al. (1974). Homologous proteins which correspond in location are in boldface type. d Figures in parentheses indicate homologous B. stearothermophilus 50S proteins according to Geisser et al. (1973). Other studies (Fahnestock et al., 1973; Tischendorf et a1., 1973) indicate that E-L2 is homologous to B-L3. e Figures in parentheses indicate homologous B. stearothermo~ philus 50S proteins according to Home and Erdmann (1972).
enables us to make a preliminary comparison of the location of tyrosine residues in some homologous ribosomal proteins from B. stearothermophilus and E. coli. In order to construct this comparison in the most valid manner, we have complied topography data for the two organisms which has been derived from iodination studies carried out under similar conditions (Table 4). Four pairs of homologous proteins (B-S7 and E-S7, B~S10 and E-S9, B-L2 and E-L2, B-L5 and E-L6) show multiple exposed tyrosines, suggesting a surface location. One pair of homologous proteins (B-S13 and E-S10) has tyrosines at the 30S:50S interface. Finally, one pair of homologous proteins (B-S9 and E-S6) is involved in conformational changes when subunits combine to form the 70S ribosome. Although the use of additional topographical probes and the examintation of more ribosomal sites will be required, it appears that tyrosines in only some of the homologous proteins from B. stearothermophihts and E. coli occupy the same position within the ribosome. One possible explanation for this unexpected finding is that tyrosines are highly variable residues in homologous ribosomal proteins from these two bacteria. There is not sufficient amino acid sequence data available at the present time to
279
either support or refute this premise. On the other hand, some homologous proteins may indeed be positioned at different loci in the ribosome. In this connection, the spatial packaging of certain key proteins could prove to be an important factor in determining the thermostability of the ribosome. Acknowledgments. This investigation was supported in part by U.S. Public Health Service grants GM 22164 (S.M.F.) and GM 19843 (C.R.C.). We thank Alice Beekman for expert technical assistance.
References Avital, S., Elson, D. : A method for changing the pH of gel strips in the two dimensional gel electrophoresis of ribosomal proteins. Analyt. Biochem. 57, 287-289 (1974) Fahnestock, S., Erdmann, V., Nomura, M.: Reconstruction of 50S ribosomal subunits from protein-free ribonucleic acid. Biochemistry 12, 220-224 (1973) Fahnestock, S., Nomura, M.: Activity of ribosomes containing 5S RNA with a chemically modified 3'-terminus. Proc. nat. Acad. Sci. (Wash.) 69, 363 365 (1972) Friedman, S.M., Axel, R., Weinstein, I.B. : Stability of ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus. J. Bact. 93, 1521-1926 (1967) Friedman, S.M., Weinstein, I.B. : Protein synthesis in a subcellular system from Bacillus stearothermophilus. Biochim. biophys. Acta (Amst.) 114, 593~505 (1966) Geisser, M., Tischendorf, G.W., Stoffler, G.: Comparative immunological and electrophoretic studies on ribosomal proteins of Bacillaceae. Molec. gen. Genet. 127, 129-145 (1973) Hardy, S.J.S., Kurland, C.G., Voynow, P., Mora, G. : The ribosomal proteins ofEscherichia coli 1. Purification of the 30S ribosomal proteins. Biochemistry 8, 2897~905 (1969) Held, W.A., Gette, W.R., Nomura, M.: Role of 16S ribosomal ribonucleic acid and the 30S ribosomal protein S12 on the initiation of natural messenger ribonucleic acid translation. Biochemistry 13, 2115 2122 (1974) Higo, K., Held, W., Kahan, L., Nomura, M.: Functional correspondence between 30S ribosomal proteins of Escherichia coli and Bacilh~s stearothermophilus. Proc. nat. Acad. Sci. (Wash.) 70, 944-948 (1973) Higo, K.I., Loertscher, K.: Amino-terminal sequences of some Escherichia coli 30S ribosomal proteins and functionally corresponding Bacillus stearothermophilus ribosomal proteins. J. Bact. 118, 180-186 (1974) Home, J.R., Erdmann, V.A.: Isolation and characterization of 5S RNA-protein complexes from Bacillus stearolhermophilus and Escherichia coli ribosomes. Molec. gen. Genet. 119, 337 344 (1972) Isono, K.: Altered 30S ribosomal proteins in streptomycin resistant, dependent and independent mutants of Bacillus stearothermophilus. Molec. gen. Genet. 133, 77 86 (1974) Isono, S., Isono, K. : Purification and characterization of 30S ribosomal proteins from Bacillus stearothermophilus. Europ. J. Biochem. 50, 483-488 (1975) Isono, K., Isono, S., Stoffler, G., Visentin, L.P,, Yaguchi, M., Matheson, A.T.: Correlation between 30S ribosomal proteins of Bacillus stearothermophilus and Escherichia coli. Molec. gen. Genet. 127, 191-195 (1973) Kaltschmidt, E., Wittmann, H.G. : Ribosomal proteins VII. Twodimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Analyt. Biochem. 36, 401-412 (1970)
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Litman, D.J., Cantor, C.R. : Surface topography of the Escherichia coli ribosome. Enzymatic iodination of the 50S subunit. Biochemistry 13, 512-518 (1974) Litman, D.J., Lee, C.C., Cantor, C.R. : Evidence for a conformational change in the 30S E. coli ribosomal subunit upon formation of 70S particles. FEBS Letters 47, 268-271 (1974) Lodish, H.F. : Species specificity of polypeptide chain initiation. Nature (Lond.) 224, 867-870 (1969) Michalski, C.J., Sells, B.H. : Molecular morphology of ribosomes. Structural considerations of Escherichia coli ribosomal subunits utilizing lactoperoxidase-catalyzed iodination of ribosomal proteins. Europ. J. Biochem. 49, 361 367 (1974) Michalski, C.J., Sells, B.H. : Molecular morphology of ribosomes. Iodination of Escherichia coli ribosomal proteins with solidstate lactoperoxidase. Europ. J. Biochem. 52, 385-389 (1975) Michalski, C.J., Sells, B.H., Morrison, M. : Molecular morphology of ribosomes. Localization of ribosomal proteins in 50-S subunits. Europ. J. Biochem. 33, 481~485 (1973) Miller, R.V., Sypherd, P.S.: Topography of the Escherichia coli 30S ribosome revealed by the modification of ribosomal proteins. J. molec. Biol. 78, 539 550 (1973) Mora, G., Donner, D., Thammana, P., Lutter, L., Kurland, C.G. : Purification and characterization of 50S ribosomal proteins of Escherichia coli. Molec. gen. Genet. 112, 229-242 (1971)
Nirenberg, M.W. : Cell-free protein synthesis directed by messenger RNA. Methods Enzymol. 6, 1723 (1963) Nomura, M. : Assembly of bacterial ribosomes. Science 179, 864~ 873 (1973) Nomura, M., Traub, P., Bechmann, H.: Hybrid 30S ribosomal particles reconstituted from components of different bacterial origins. Nature (Lond.) 219, 793-799 (1968) Tischendorf, G.W,, Geisser, M., Stoffler, G. : Comparison of ribosomal RNA-binding protein "L2" isolated from different bacterial species, Molec. gen. Genet. 127, 147-156 (1973) Visentin, L.P., Matheson, A.T., Yaguchi, M. : Homologies in procaryotic ribosomal proteins: alanine rich acidic proteins associated with polypeptide translocation. FEBS Letters 41, 310-313 (1974) Yaguchi, M., Matheson, A.T., Visentin, L.P.: Procaryotic ribosomal proteins: N-terminal sequence homologies and structural correspondence of 30S ribosomal proteins from Escherichia coli and Bacillus stearothermophilus. FEBS Letters 46, 296-300 (1974)
Communicated by H.G. Wittmann Received December 22, 1975
Surface Topography of the Bacillus stearothermophilus Ribosome Hugh M. Miller and S. Marvin Friedman Department of Biological Sciences, Hunter College of the City University of New York, New York, N.Y., U.S.A.
David J. Litman and Charles R. Cantor Departments of Chemistry and Biological Sciences, Columbia University, New York, N.Y., U.S,A.
Summary. The surface topography of the intact 70S ribosome and free 30S and 50S subunits from Bacillus stearothermophilus strain 2184 was investigated by lactoperoxidase-catalyzed iodination. Two-dimensional polyacrylamide gel electrophoresis was employed to separate ribosomal proteins for analysis of their reactivity. Free 50S subunits incorporated about 18% more 125I than did 50S subunits derived from 70S ribosomes, whereas free 30S subunits and 30S subunits derived from 70S ribosomes incorporated similar amounts of 125I. Iodinated 70S ribosomes and subunits retained 62-78% of the protein synthesis activity of untreated particles and sedimentation profiles showed no gross conformational changes due to iodination. The proteins most reactive to enzymatic iodination were $4, $7, S10 and Sa of the small subunit and L2, L4, L5/9, L6 and L36 of the large subunit. Proteins $2, $3, $7, S13, Sa, L5/9, L10, Lll and L24/25 were labeled substantially more in the free subunits than in the 70S ribosome. Other proteins, including $5, $9, S12, S15/16, S18 and L36 were more extensively iodinated in the 70S ribosome than in the free subunits. The locations of tyrosine residues in some homologus ribosomal proteins from B. stearothermophilus and E. coli are compared.
Introduction
Ribosomes from the thermophilic bacterium, Bacillus stearothermophilus, are of particular interest because they differ from Escherichia coli ribosomes in several important properties, including heat stability (Friedman et al., 1967) and the translation of polycistronic messenger RNA's (Lodish, 1969). Both protein S12 and 16S RNA have recently been implicated in the failure of thermophile ribosomes to translate the coat protein and replicase cistrons of bacteriophage R17
RNA (Held et al., 1974). Although the molecular basis for the unusual heat stability of thermophile ribosomes remains unknown, reconstitution studies with mixtures of E. coli and thermophile ribosomal components revealed that B. stearothermophilus ribosomal proteins play a key role in protecting the ribosome from thermal denaturation (Nomura et al., 1968). Experiments employing reconstruction, immunological, chemical and genetic approaches have established correspondence between many E. coli and B. stearothermophilus ribosomal proteins (Horne and Erdmann, 1972; Fahnenstock et al., 1973; Geisser et al., 1973; Isono et al., 1973; Tischendorf et al., 1973; Higo and Loertscher, 1974; Isono, 1974; Visentin et al., 1974; Yaguchi et al., 1974). Since lactoperoxidase-catalyzed iodination has proven to be a valuable technique for probing the topography of E. coli ribosomal proteins (Michalski et al., 1973; Miller and Sypherd, 1973; Litman and Cantor, 1974; Litman et al., 1974; Michalski and Sells, 1974, 1975), it was of interest to apply this method also to thermophile ribosomes. One would then be in a position to ask if tyrosines in homologous ribosomal proteins from these different bacterial species occupy similar sites within the ribosome. In addition, information concerning the structural organization of thermophile ribosomal proteins could lead to a clearer understanding of ribosome stability. In the present report, we have utilized enzymatic iodination to study the surface topography of the B. stearothermophilus 70S ribosome and isolated subunits. The results are interpreted in regard to proteins with predominantly buried and exposed tyrosine residues, proteins involved in the 30S:50S interface region and conformational changes in proteins attendant on subunit interaction. A comparison of these classifications for some homologous ribosomal proteins from B. stearothermophilus and E. coli is presented.
274
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
Materials and Methods Buffers Buffers of the following composition were used in this study: TMAI (0.01 M Tris-HC1, pH 7.5, 0.01 M Mg acetate, 0.03 M NH4C1, 0.006 M 2-mercaptoethanol); TMA-II (0.01 M Tris-HC1, pH 7.5, 0.0003 M Mg acetate, 0.03 M NH4C1, 0.006 M 2-mercaptoethanol); TMN-II (0.01 M Tris-HC1, pH 7.5, 0.001 M Mg acetate, 0.1 M NH4C1, 0.006 M 2-mercaptoethanol).
Preparation of Ribosomes B. stearothermophilus strain 2184 was grown at 65 ° C to midlogarithmic phase and the S-30 fraction was prepared as previously described (Friedman and Weinstein, 1966). All of the following procedures were carried out at 4 ° C. The S-30 fraction was layered onto a buffered 1.1 M sucrose-0.5 M NH4C1 solution (Fahnestock and Nomura, 1972) in the ratio of 1 volume S-30 to 3 volumes sucrose. Ribosomal particles were pelleted by centrifugation at 28,000 rpm for 16 h in a Spinco 42.1 rotor and resuspended in TMA-I buffer. 70S ribosomes were purified by centrifugation on 7-30% linear sucrose gradients in TMA-I buffer at 20,000 rpm for 16 h in a Spinco SW 25.1 rotor. Fractions (1 ml) were collected by withdrawing the contents from the bottom of each tube and only those fractions containing 70S ribosomes free of 50S subunits were pooled. Pure 70S ribosomes were pelleted and resuspended in TMA-I buffer at a concentration of approximately 20 mg/ml. 70S ribosomes were dissociated by dialysis against TMN-II buffer for 40 h and the subunits were separated on 10-25% discontinuous sucrose gradients centrifuged in a Spinco SW 25.1 rotor at 22,000 rpm for 16 h. Pure subunits were obtained by aspirating the 30S subunit band located near the middle of the tube and fractionating the more diffuse 50S subunit band located toward the bottom of the tube. When small amounts of subunits were to be separated from iodinated 70S ribosomes, a 7-25% linear sucrose gradient was centrifuged in a Spinco SW 27 rotor at 20,000 rpm for 17 h. Analytical gradients (5-20% sucrose) were centrifuged in a Spinco SW 50.1 rotor at 45,000 rpm for 90 min.
Iodination of Ribosomes 70S, 30S and 50S particles were dialyzed against TMA-I buffer minus 2-mercaptoethanol and iodinated according to the procedure of Litman and Cantor (1974). The specific activity of the Na 125I used was 500 mCi/mM (New England Nuclear). Typical reactions contained t0 mg of 70S ribosomes, 4 mg of 30S subunits or 8 mg of 50S subunits. After the reaction was stopped by the addition of 2-mercaptoethanol, the particles were pelleted, resuspended and dialyzed against TMA-II buffer. Iodinated 70S ribosomes were separated into 30S and 50S subunits as described above. Unlabelled subunits were added to these derived subunits and to iodinated free 30S and 50S subunits to yield final concentrations of 17 mg 30S subunits and 28 mg 50S subunits.
Extraction of Ribosomal Proteins 30S and 50S subunits were stripped of protein by the acetic acid method of Hardy et al. (1969).
Two-dimensional Gel Electrophoresis and Sample Counting Two-dimensional gel electrophoresis of ribosomal proteins was carried out according to Kaltschmidt and Wittmann (1970) except
that the first dimension gel was immersed in 0.3 M HC1 for 5 min in order to effect the pH change (Avital and Elson, 1974). After staining the gel slabs in 1% Amido Black and 7% acetic acid and destaining with 7% acetic acid, the protein spots were carefully cut out with a scalpel and placed in scintillation vials. The samples were treated and counted as previously described (Litman and Cantor, 1974).
Protein Synthesis Assay The activity of ribosomes and subunits was assayed by poly Udirected phenylalanine incorporation (Nirenberg, t963) under conditions of limiting ribosomes as described by Litman and Cantor (1974).
Results
Iodination of Ribosomal Particles 70S, 30S and 50S ribosomal particles were iodinated to less than saturation levels in parallel reaction mixtures according to the procedure of Litman and Cantor (1974). The iodinated 70S ribosomes were then dissociated and pure 30S and 50S subunits were isolated by sucrose gradient sedimentation. The specific activities of these subunits were compared to those of subunits iodinated in the free state. Iodinated free 30S subunits and 30S subunits derived from iodinated 70S ribosomes had almost identical iodine contents of 22.1 and 22.0 iodines per particle, respectively. The free 50S subunit incorporated 29.6 iodines per particle, whereas the 50S subunit derived from iodinated 70S ribosomes incorporated 25.1 iodines per particle. Thus, the free 50S subunit incorporated about 18% more 125I than did the 50S moiety of the 70S ribosome. Similar labeling data were reported by Litman and Cantor (1974) for the E. coli 50S subunit.
Effect of lodination on Ribosome Activity and Sedimentation Properties In order to obtain meaningful structural information for ribosomes from experiments employing a topographical probe, the chemical modification should not significantly alter the functional activity of the particle nor cause it to undergo extensive conformational changes. Therefore, the protein synthesis activities of iodinated and unlabeled 70S, 30S and 50S particles were compared. Ribosomes and subunits were iodinated to the same extent as those which were used for two-dimensional gel electrophoresis of ribosomal proteins except that low specific activity (1 mCi/mM) 12sI was used. The intrinsic trichloroacetic acid precipitable counts, which never amounted
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
to more than 5% of the total counts, were substracted from the counts obtained in the protein synthesis assay. Table 1 shows that 70S particles retained 79% of the control protein synthesis activity after they had been heavily labeled (40 iodines/particle). 30S subunits labeled to the extent of 22 iodines/particle retained 62% of the control activity and 50S subunits labeled to the extent of 29 iodines/particle retained 72% of the control activity. Figures 1A and B show sedimentation profiles of iodinated and unlabeled 30S and 50S subunits. The sedimentation velocities were found to be virtually identical for both iodinated and unlabeled particles, indicating that no gross conformational distortion had occurred as a result of the covalent reaction.
Approximately two milligrams of ribosomal proteins were used for each two-dimensional gel separation. Figures 2A and B show typical two-dimensional gel electrophoretic patterns of 30S and 50S subunit proteins from B. stearothermophilus strain 2184. The 30S pattern of 22 isolated proteins was virtually identical to that obtained by Isono and Isono (1975) for B. stearothermophilus strain 799 and their numbering system was employed to identify the spots. In the case of 50S pattern, most of the 44 proteins corresponded to the 38 proteins found in strain 799 by Horne and Erdmann (1973) and their numbering system was employed to identify the matching spots. The six extra proteins present in strain 2184 but absent in strain 799 were numbered LX1 through LX6. Spots corresponding to proteins S1 and LX4 were often too faint to observe and could not always be cut out of gels. These proteins may be only weakly associated with ribosomes and thus partially removed during the isolation procedure as has been shown
Table 1. Effect of iodination on the protein synthesis activity of B. stearothermophilus ribosomal particles
70S 70S 30S 30S 50S 50S
Iodines/ particle
0 47 0 22 0 29
!.6
4.0
A260
0.8
_.jl 4
f
cpmxlO 3 2.0
8 12 16 fraction number
20
S 2.5
2.0
cpm x 103,
A26o
Identification of Iodinated Ribosomal Proteins
Particle
275
Activity a Poly U-phe incorp. (cpm x 105/mg particle)
Per cent
3.9 3.1 7.8 4.8 5.6 4.1
100.0 78.8 100.0 62.0 100.0 72.0
a Assay conditions for poly U-directed phenylalanine incorporation are referred to in Materials and Methods.
1.2
1.5
0.4
0.5 J t
4
8
I
12 fraction
16
number
20
Fig. 1 A and B. Sucrose gradient sedimentation profiles of untreated (triangles) and iodinated (circles) (A) 30S and (B) 50S subunits from B. stearothermophilus
to be the case for E. coli protein S1 (Nomura, 1973). Several protein spots were not sufficiently resolved to be cut from the gels as individual proteins and were therefore excised as pairs or triads. Proteins treated in this manner were S15/16, L5/9, L7/8, L12/ 14/15, L16/19/20, L24/25, L30/31, L34/35 and L37/38. Table 2 shows the radioactivity data obtained for proteins that were labeled in isolated 30S subunits and in 30S subunits derived from labeled 70S ribosomes. Table 3 shows the radioactivity data obtained for proteins labeled in isolated 50S subunits and in 50S subunits derived from labeled 70S ribosomes. The values presented are the averages from six individual gels in each case. The radioactivity of each protein has been corrected for the total amount of protein that actually ran on to the gel as well as for the specific activity of each subunit according to the calculations described by Litman and Cantor (1974). The radioactivity data presented in Tables 2 and 3 has been processed two ways. One calculation expressed the difference between the extent of iodination (expressed as tyrosine equivalents) of each protein in isolated subunits and the extent of iodination of each protein in subunits derived from iodinated 70S ribosomes. A second calculation expresses the
276
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome Fig. 2A and B. Two-dimensional electrophoretic patterns of B. stearothermophilus strain 2184 (A) 30S and (B) 50S ribosomal proteins
ratio of the extent of iodination (expressed as tyrosine equivalents) of each protein in isolated subunits to the extent of iodination of each protein in subunits derived from iodinated 70S ribosomes. Only proteins which have been substatially iodinated (> 800 cpm) have been treated in this manner in order to eliminate values which may not be statistically significant. It can be seen from Tables 2 and 3 that the extent of iodination varies considerably for individual proteins in each subunit. 30S proteins that have been extensively labeled include $4, $7, S10, S15/16 and Sa, whereas 50S proteins that have been extensively labeled include L2, L4, L5/9, L36. These proteins presumably have several tyrosines exposed on the surface of the ribosome and are thus readily accessible
to lactoperoxidase-catalyzed iodination. On the other hand, proteins that have been labeled to a small extent in 30S and 50S subunits include S14, $20, L3, L7/8, L12/14/15, L13, L27, L37/38, LXs and LX 6. These proteins must be unreactive to enzymatic iodination either because their tyrosine content is low or their tyrosines are buried deep within the ribosome structure. Heavily iodinated proteins that have been labeled more in isolated 30S subunits than in 30S subunits derived from 70S ribosomes are $3, $4, $7, S13 and Sa, whereas those that have been labeled more in isolated 50S subunits than in 50S subunits derived from 70S ribosomes are L2, L5/9, L10, L l l , L24/25 and LX1. Since these proteins are less reactive to
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome
enzymatic iodination in the intact 70S ribosome than in the free subunits, they may be associated with the 30S:50S interface region. Heavily iodinated proteins that have been labeled more in subunits derived from 70S ribosomes than in isolated subunits are $5, $9, S12, S15/16, S18 and L36. These results indicate that conformational changes occur in or around these proteins when subunits associate to form the 70S ribosome.
Discussion
Lactoperoxidase-catalyzed iodination is generally considered to be a probe of surface topography because the large size of the enzyme (M.W. 78,000) would preclude its ability to penetrate the ribosome structure. Support for this contention comes from the iodination data reported by Litman and Cantor (1974) for the E. coli 50S ribosomal proteins. They, demonstrated that heavily labeled proteins did not constitute a specially selected class of tyrosine-rich proteins. It should be noted that in the case of the present results for B. stearothermophilus, there also does not appear to be any correlation between the extent of iodination and the tyrosine content of the
277
ribosomal proteins. Since the quantitative amino acid composition of isolated ribosomal proteins from B. stearothet~nophilus strain 2184 has not yet been determined, such a comparison must be based upon the quantitative amino acid data reported by Isono and Isono (1974) for the 30S ribosomal proteins from B. stearothermophilus strain 799. Their analysis showed, for example, that B-S19 had a high tyrosine content, whereas no tyrosine was detected in B-S10. In the present study, we found that B-S19 was labeled to a very small degree, whereas B-S10 was labeled to a relatively large degree. The extensive labeling of B-S10, presumably a tyrosine-free protein, was at first difficult to interpret. However, a careful examination of the sequence data (Yaguchi et al., 1974) for the first 15 amino acids starting at the N-terminal position of B-S10 derived from B. stearothermophih~s strain 10 revealed the presence of two tyrosine residues. It remains to be seen whether these variations are due to differences in the analytical techniques employed or in the amino acid composition of homologous proteins from different strains of B. stearothermophilus. The absolute counts found in a protein spot will be limited by the number of tyrosine residues present in that particular protein. This can best be illustrated
Table 2. 1251 Labelling of B. stearothermophilus 30S ribosomal proteins Protein
B-S1 B-S2 B-S3 B-S4 B-S5 B-S6 B-S7 B-S8 B-S9 B-S10 B-S11 B-S12 B-S13 B-S14 B-S15/16 B-S17 B-S 18 B-S19 B-S20 B-S21 B-Sa
cpm 30S ( x 10 -2)
11.8 12.8 46.2 70.9 35.2 39.7 343.1 40.5 20.0 81.4 8.2 36.8 21.6 1.4 81.5 6.8 45.9 9.3 1.2 4.9 74.3
Tyr. Equiv.
0.26 0.28 1.03 1.58 0.78 0.88 7.62 0.90 0.44 1.81 0.18 0.82 0.48 0.03 1.81 0.15 1.02 0.21 0.03 0.10 1.65
cpm 30S via 70S ~ ( x 10 -2) 12.8 5.4 36.2 61.4 58.9 34.6 278.2 45.6 33.8 77.9 10.6 51.0 9.2 1.9 129.6 15.2 63.8 9.4 1.4 4.0 60.7
Tyr. Equiv.
0.28 0.12 0.80 1.36 1.31 0.77 6.18 1.01 0.75 1.73 0.24 1.13 0.20 0.04 2.88 0.38 1.42 0.21 0.03 0.09 1.35
ATyr. b
30S 30S via 70S
-0.16 0.23 0.22 -0.53 0.11 1.44 -0.11 -0.31 ---0.31 0.28 . . -1.07 -0.23 - 0.40 -. . . . 0.30
2.33 1.29 0.60 1.23 -0.59 0.75 0.73 2.40 .
.
. 0.63 0.39 0.72 --
. .
. .
. . 1.22
Iodines/30S=22.1 ; Iodines/30S via 70S=22.0. b c
30S subunits isolated from iodinated 70S ribosomes. (30S--30S via 70S) tyrosine values less than 0.1 are denoted by - - . Tyrosine ratios within the range of 0 . 8 - 1.2 are denoted by - - ; ratios for proteins with low levels of labeling are denoted by .... .
H . M . Miller et al: Topography of the
278
Bacillus stearothermophilus Ribosome
Table 3. 1251 Labelling of B. stearothermophilus 50S ribosomal proteins Protein
cpm 50S ( x 10- z)
Tyr.
c p m 50S
Equiv.
via 70S a
Tyr. Equiv.
ATyr. b
50S 50S via 70S
0.38 1.74 0.05 1.40 9.15 1.20 0.04 0.37 0.53 0.03 0.01 2.22 0.55 0.28 0.09 0.10 0.59 0.31 0.19 0.04 0.65 0.11 0.08 0.06 0.03 0.33 2.33 0.04 0.36 0.23 0.19 0.05 0.04
0,13 0.21 . . 0.14 3.35 0,19 . . 0.29 0.27 . . . . ---. . . . -0.43 0.10 . . 0.17 ----
1.34 --
( x 10 -2) B-L1 B-L2 B-L3 B-L4 B-L5/9 B-L6 B-L7/8 B-L10 B-Lll B-L12/14/15 B-L13 B-L16/19/20 B-L17 B-L18 B-L21 B-L22 B-L23 B-L24/25 B-L26 B-L27 B-L28 B-L29 B-L30/31 B-L32 B-L33 B-L34/35 B-L36 B-L37/38 B-LX 1 B-LX2 B-LX 3 B-LX5 B-LX 6
21.4 82.1 3.5 64.9 528.4 58.7 2.8 27.7 33.8 2.0 1.0 89.7 26.5 13.2 5.1 6.3 27.4 31.0 12.3 1.7 34.6 5.0 5.0 4.0 2.3 14.9 81.9 1.7 24.3 13.9 14.4 2.8 2.3
0,51 1,95 0.08 1.54 12.50 1.39 0.07 0.66 0.80 0.05 0.02 2.13 0.63 0.3• 0.12 0.15 0.65 0.74 0.29 0.04 0.82 0.12 0.12 0.10 0.05 0.35 1.94 0.04 0.58 0.33 0.34 0.07 0.06
16.2 73.3 2.2 59.2 385.9 50.4 1.5 15.4 22.4 1.1 0.5 93.8 23.2 11.9 3.8 4.4 24.7 13.2 7.9 1.7 27.6 4.5 3.3 2.5 1.2 13.8 98.1 1.7 15.0 9.6 8.0 0.5 0.4
. . 0.39 . . 0.22 0.10 0.15 . . . .
.
.
. -1.37 --
.
.
. 1.78 1.51
. .
. .
. . ----
. .
. .
. . -2.39 1.53
.
. 1.26 - --....
.
.
.
.
.
.
0.83 1.61 1.43 1.79 . .
. .
. .
I o d i n e s / 5 0 S = 2 9 . 6 ; Iodines/50S via 7 0 S = 2 5 . 1 , a b c
50S subunits isolated from iodinated 70S ribosomes. ( 5 0 S - 50S via 70S) tyrosine values less than 0.1 are denoted by - - .
Tyrosine ratios within the range of 0.8-1.2 are denoted by - - ;
ratios for proteins with low levels of labeling are denoted by .... .
in the extreme case of proteins which have been shown to be devoid of tyrosine, namely E-L7 and E-L12 (Mora et al., 1971) and B-L7 and B-L12 (Visentin et al., 1974). Litman and Cantor (1974) found that very few counts were incorporated into E-L7 and E-L12, and very low labeling levels were also observed for B-L7 and B-L12 in the present study. The topography of these important ribosomal proteins cannot be revealed by enzymatic iodination and will have to be determined by other techniques. The results obtained from the present experiments provide evidence for the structural organization of proteins within the B. stearothermophilus ribosome. Heavily labeled proteins, notably $4, $7, S10, Sa, L2, L5/9, L6 and L36, are likely to have a large fraction of their tyrosine residues exposed on the surface of the ribosome. Proteins that were labeled more
extensively in isolated subunits than in the intact 70S ribosome include $2, $3, $7, S13, Sa, L5/9, L10, Lll and L24/25. These proteins are either associated with the 30S:50S interface region or are affected by conformational changes when subunits interact resulting in the masking of some tyrosine residues. Proteins that were more heavily labeled in the intact 70S ribosome than in the isolated subunits include $5, $9, S12, S 15/16, S 18 and L36. This suggests that these proteins undergo or are affected by conformational changes when subunits combine resulting in enhanced exposure of some tyrosine residues. Such a relaxation or expansion of ribosomal structure is particularly evident in the case of the 30S subunit. A similar effect has also been observed for the E. coli 30S subunit by Litman et al. (1974). The availability of the data detailed above now
H.M. Miller et al: Topography of the Bacillus stearothermophilus Ribosome Table 4.Topography of B. stearothermophilus and E. coli ribosomal proteins as determined by enzymatic iodination Location
B. stearothermophilus "
E. coli b
Highly exposed
$4, $7, S10, Sa
$5(6) c, $7(7), $9(10), S18(19)
L2, L4, L5/9, L6, L36
L2(2) d, L5, L6(5) e, L10(10) d, L11, L26
30S:50S Interface
$2, $3, $7, S13, Sa
$10(13)
L5/9, L10, L l l , L24/25
L2, L13(13) a, L18(22) ~, L26, L27, L28
Conformational changes upon 70S formation
$5, $9, S12, S15]16, S18
$3, $6(9), $9, S 18(19)
L36
Lll
" Data from the present study. b Data of Litman et al. (1974) for the 30S proteins and of Litman and Cantor (1974) for the 50S proteins. c Figures in parentheses indicate homologous B. stearothermophilus 30S proteins according to Yaguchi et al. (1974). Homologous proteins which correspond in location are in boldface type. d Figures in parentheses indicate homologous B. stearothermophilus 50S proteins according to Geisser et al. (1973). Other studies (Fahnestock et al., 1973; Tischendorf et a1., 1973) indicate that E-L2 is homologous to B-L3. e Figures in parentheses indicate homologous B. stearothermo~ philus 50S proteins according to Home and Erdmann (1972).
enables us to make a preliminary comparison of the location of tyrosine residues in some homologous ribosomal proteins from B. stearothermophilus and E. coli. In order to construct this comparison in the most valid manner, we have complied topography data for the two organisms which has been derived from iodination studies carried out under similar conditions (Table 4). Four pairs of homologous proteins (B-S7 and E-S7, B~S10 and E-S9, B-L2 and E-L2, B-L5 and E-L6) show multiple exposed tyrosines, suggesting a surface location. One pair of homologous proteins (B-S13 and E-S10) has tyrosines at the 30S:50S interface. Finally, one pair of homologous proteins (B-S9 and E-S6) is involved in conformational changes when subunits combine to form the 70S ribosome. Although the use of additional topographical probes and the examintation of more ribosomal sites will be required, it appears that tyrosines in only some of the homologous proteins from B. stearothermophihts and E. coli occupy the same position within the ribosome. One possible explanation for this unexpected finding is that tyrosines are highly variable residues in homologous ribosomal proteins from these two bacteria. There is not sufficient amino acid sequence data available at the present time to
279
either support or refute this premise. On the other hand, some homologous proteins may indeed be positioned at different loci in the ribosome. In this connection, the spatial packaging of certain key proteins could prove to be an important factor in determining the thermostability of the ribosome. Acknowledgments. This investigation was supported in part by U.S. Public Health Service grants GM 22164 (S.M.F.) and GM 19843 (C.R.C.). We thank Alice Beekman for expert technical assistance.
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Communicated by H.G. Wittmann Received December 22, 1975
