[73]
RIBOSOMAL FUNCTION&L SITES
621
[73] Affinity Labeling of Ribosomal Functional Sites By ADA ZAMIR Ribosomes perform multiple functions in the course of protein synthesis; recognition and binding of the initiation region of mRNA, decoding of mRNA by binding of cognate aminoacyl-tRNAs, catalysis of peptide bond formation, and the translocation of nascent peptidyl-tRNA and mRNA. In addition, ribosomes can specifically bind a number of antibiotic compounds that interfere with one or another facet of protein biosynthesis. This diversity of function is matched by the complexity of ribosomal structure, an intricate arrangement of protein and RNA molecules, whose highly cooperative interaction is responsible for the formation of ribosomal functional sites. 1 This unique feature of the ribosome greatly obstructs the identification of ribosomal components directly located at specific active centers, and it renders difficult the distinction between directly and indirectly involved components. One approach that in principle circumvents this difficulty is based on the method of affinity labeling. Employing to date several classes of chemically or photoreactive site-specific ligands, this method has been applied to identify ribosomal components located within close range of several functional sites on the Escherichia coli ribosome. This chapter reviews methods applied in affinity labeling studies of ribosomes and describes the sites under study, major types of ligand used, criteria for establishing the specificity of labeling, and the characterization of modified ribosomal components. It does not include procedures for the synthesis of affinity labeling probes or a detailed discussion of the results obtained in the different studies. These can be found in the cited publications or elsewhere in this volume. For a general consideration of particular problems related to affinity labeling of ribosomes, see Cantor et al? and this volume [][5]. Early experiments are reviewed by Pongs et al2
mRNA Analogs Site Studied. In the normal functioning of the ribosome, mRNA initially binds to the ribosome in a manner allowing translation to start 1 H. G. Wittmann, Eur. d. Biochem. 61, 1 (1976). 2 C. R. Cantor, M. Pellegrini, and H. Oen, in "Ribosomes" (M. Nomura, A. Tissi~res, and P. Lengyel, eds.), p. 573. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. See also this volume [15]. 8O. Pongs, K. H. Nierhaus, V. A. Erdmann, and H. G. Wittmann, FEBS Lett. 40 (suppl.), 28 (1974).
622
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
at the correct initiation codon. Initiation and subsequent codons will then be decoded by selection and binding to the ribosome of the respective cognate aminoacyl-tRNAs. This process is actively affected by the ribosome. 4 Affinity labeling studies of the decoding site (defined as the ribosomal site in contact with the codon to be decoded) are based on the existence of relatively simple model compounds for mRNA. These include short oligonueleotides regarded as single isolated codons, or homopolynueleotides such as poly(U). Comparable to codons in natural mRNA, such analogs can direct binding of cognate aminoacyl-tRNAs to the ribosome and can, therefore, be considered to bind at the ribosomal decoding site. Two such sites may exist on the ribosome corresponding to the donor and acceptor positions of tRNA derivatives. While the initiation triplet is likely to direct binding to the donor site, all other triplets are decoded at the acceptor site. Reagents. Table I specifies all mRNA analogs reported to date as affinity labeling probes for ribosomal decoding sites. T M The table lists a group of oligonucleotides of defined length and sequence where chemically reactive groups have been introduced as substituents of the phosphate (a-c), pyrimidine ring (d-f), or ribose (g) groupings. Whereas the haloacetyl group in compounds a-f will make likely a reaction with nucleophilic groups on ribosomal proteins, reagent g was designed to interact with G residues in the rRNA. In addition, the list includes poly (U) and poly (4-thio-U), both of which are photoreactable. Oligonucleotides, Tests o] Function
The binding and coding properties of modified oligonucleotides were examined in order to establish their adequacy as affinity labeling probes. ' L. Gorini, Nature (London), New Biol. 234, 261 (1971). 50. Pongs and E. Lanka, Proc. Natl. Acad. Sci. U~S.A. 72, 1505 (1975). 60. Pongs, G. StSffier,and E. Lanka, J. Mol. Biol. 99, 301 (1975). O. Pongs and E. Rossner, Nucleic Acids Res. 3, 1625 (1976). s j. Petre and D. Elson (1976). Submitted for publication. "R. Liihrmann,U. Schwarz, and H. G. Gassen,FEB8 Lett. 32, 55 (1973). x0R. Lfihrmann,H. G. Gassen, and G. StSffier,Eur. J. Biochem. 66, 1 (1976). n O. Pongs, G. StSffler,and R. W. Bald, Nucleic Acids Res. 3, 1635 (197~). n R. Wagner and H. G. Gassen, Biochem. Biophys. Res. Cammun. ~ $19 (1975). 11I. Fiser, K. It. Seheit, G. StSffier, and E. Kueehler; /~o~'hem. ~ y s . Res. Commun. 601 1112 (1974). 1~E. ~mehl~r,. A. Ba~ta, L Fiser,, tL i~au~ma~m,, F. l~argaritella, W. Maurer, and G. ~6]~er;,A b s ~ In~. C~ngr. B~och~m. lOth, l~,ambur~03-3-153 (1976). 15M. L. Schenkman, D. C: Wards and P. B. Moore, Biochim. Biophys. Acta 353, 503 (1974). I. Fiser, P. Margaritella, and E. Kuechler,FEBS Left. 521 281 (1975).
[73]
623
RIBOSOMAL FUNCTIONAL SITES
TABLE I mRNA ANALOGS Reagent"
Modified group
?, (a) p*ApUpG
(b) p*UpGpA
(c) p*ApApA
0~~NH--
References
?,
0 tl HO--~--O-~ O~NH--C--CH~Br
O tl HO--P--O~--. I
/f-'~\
HN-~~Ntt-O
6
C--CH~Br 7
O
II
C-- CI~ Br
8
O
(d) UpUpUpU*
l O
O
(e) GpUpUpU*
I O (f) ApUpGpU*
O
O~N.!
11
I O
(g) UpUpUpU**
CH~O--
12
OO HN OH tt H /~----h J H--C--C~C----O (h) Poly(4-thio-U)
13, 14
(i) Poly(U)
15, 16
a Asterisks indicate modified component.
624
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
Although these tests are best performed in the absence of covalent binding, in most cases no care has been taken to avoid irreversible binding during the assay. Thus, for example, GpUpUpU ~ has been shown to direct the binding of Val-tRNA to an even greater extent than the nonmodified oligonucleotide,TM possibly reflecting a stabilization due to the irreversible binding of template. However, other oligonucleotide probes did not exhibit such enhanced activity, e'g" 11 Another criterion for functionality of short oligonucleotide templates is the stimulation of their binding to ribosomes by cognate aminoacyltRNAs. 8 Labeling Reaction. Labeling media were buffered at pH 7.2 to 7.4 and usually contained: 6-20 mM Mg 2÷, 80-150 mM NH4C1, and radioactively labeled affinity reagent at 10- to 100-fold excess over ribosomes added in the 70 S form or as isolated 30 S subunits (sometimes preactivated17). To enhance the specificity and stability of the reversible complex of oligonucleotide and ribosome, some reaction mixtures were supplemented with the cognate aminoacyl-tRNAs. 8,~°,12 Labeling mixtures were usually incubated for 1-2 hr at 0°-37% Maximal labeling of 30 S subunits or 70 S ribosomes by the various reagents ranged between 0.1 and 0.7 mole of reagent per mole of ribosome. However, the routine determination of reagent uptake by Millipore filtration does not appear to be reliable, since the values measured by this method by far exceed the recovery of label after partial fractionation2 The efficiency and pattern of labeling are markedly affected by the state of the ribosomes. Differences in reaction pattern were noted with 70 S ribosomes as compared to 30 S subunits, 6 with freshly prepared and stored 70 S ribosomes, 6 and with crude and salt-washed 70 S ribosomes or 30 S subunits. 1~ Particularly striking are the changes observed in the labeling of 30 S subunits with p~ApUpG, where freezing and thawing of the reaction mixture greatly enhanced uptake by making available new labeling sites on the ribosome2 Specificity o] Labeling. Several tests have been performed to determine whether affinity labeling probes were attached at functionally significant sites on the ribosome. The most crucial examination of the specificity of labeling is based on the ability of the modified ribosomes to bind the tRNA specified by the covalently bound oligonucleotide in the absence of any free template. Such tests were performed, for example, with p~ApUpG-modified 30 S subunits where IF-2 stimulated binding of fMet-tRNA~ et was demon1TA, Zamir, R. Miskin, and D. Elson, J. Mol. Biol. 60, 347 (1971).
[73]
RIBOSOMAL FUNCTIONAL SITES
625
strafed, 6 and with GpUpUpU~-modified 70 S ribosomes that were shown to be capable of binding Val-tRNA. 1° These results indicate that at least part of the modification in these cases has occurred at genuine decoding sites. Another criterion for specificity is based on the stimulation of covalent binding by cognate aminoacyl-tRNAs, s,l~ Reduced covalent reaction to ribosomes precomplexed with poly(U) and Phe-tRNA 1° and competition by nonmodified oligonucleotides 5,8 have also been taken as indications for the specificity of labeling. As indicated above, it is possible that codons bind to ribosomes at two positions corresponding to donor and acceptor sites. The nature of the site occupied by attached oligonucleotides has only been tentatively determined by testing the reactivity in the puromycin reaction of the aminoacyl-tRNA bound to the modified ribosomes2 ,1° In another case, the ability of ApUpGpU~-modified 70 S ribosomes to bind Met-tRNA~ et in the presence of EF-Tu has been considered indicative of acceptor site location of the bound codon. ~1
Polynucleotides Very little use has been made so far of polynucleotides as affinity labeling probes. The only reported cases are poly (U)15,16 and its analog poly (4-thio-U).13,14 Both can be made to react with ribosomes by irradiation at 253 nm and 335 nm, respectively. The adequacy of poly (4-thio-U) to serve as probe was indicated by its ability to direct Phe-polymerization. TM In both cases irradiated mixtures contained Phe-tRNA in addition to 70 S ribosomes and polynucleotide. As cross-linking of mRNA to the ribosome will probably arrest translocation, loss of polymerizing activity has been taken to indicate the specificity of binding. 18 It is worth noting that the average chain length of the polynucleotide probe is likely to affect the course of the reaction, as the use of long poly(U) chains has been reported to result in the formation of large cross-linked ribonucleoprotein aggregates. ~6 Analysis o] Modified Ribosomal Components. As a general rule in affinity labeling of ribosomes, excess of reagent was removed from the labeled ribosomes before any fractionation was attempted, to avoid any reaction from taking place after the structure of the ribosome had been disrupted. In cases in which 70 S ribosomes were labeled, the first step was normally the dissociation into subunits and determination of label u W. B~hr, P. Faerber, and K. H. Scheit, Eur. J. Biochem. 33, 535 (1973).
626
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
distribution in the two subunits, and subsequently in separated protein and RNA fractions. All mRNA analogs listed in Table I modified specifically the 30 S subunit, and all but compound g, which modified 16 S RNA, reacted with proteins. Modification with oligonucleotides introduces some difficulty in the identification of the labeled proteins. The attachment of a highly charged oligonucleotide probe or polynucleotide fragment alters considerably the net charge of the labeled protein and hence its electrophoretic mobility. This makes unreliable the identification of proteins by their position on the map obtained by 2-dimensional gel electrophoresis. ~,s This problem has been considered in the design of compound c, where, after the labeling reaction, the bulk of the oligonucleotide can be removed by a mild acid hydrolysis of the phosphoamide linkage2 In the other cases, partial splitting of proteins from 30 S subunits by cesium chloride gradient centrifugation6 and application of various one-dimensional gel electrophoretie techniques (quoted in references cited in footnotes 6, 10, 11) allowed a characterization of modified proteins. The most reliable and sensitive techniques for identifying the oligonucleotide-labeled proteins employ antibodies raised against purified individual ribosomal proteins. 19 Techniques used were radioimmunodiffusion, 6,1° and sucrose gradient centrifugation of soluble antigen-antibody complexesJ 8
Peptidyl-tRNA and tRNA Derivatives Sites Studied. Interactions of tRNA with the ribosome can be broadly classified into two types. (a) Interactions with the peptidyltransferase center of the 50 S ribosomal subunit involving the 3' terminus of peptidylor aminoacyl-tRNA occupying, respectively, the donor (peptidyl) or acceptor (aminoacyl) site of the catalytic center. (b) Interactions likely to involve internal parts of the tRNA molecule and designed to align and stabilize the binding of cognate tRNAs to the ribosome. Affinity labeling probes for mapping of ribosomal components involved in interactions of the first type were constructed by introducing suitable substituents at the free terminal amino group of aminoacyl or peptidyl tRNA. To study the second class interactions, sites within the tRNA molecule must be rendered capable of reacting with potential binding sites on the ribosome. Most studies to date have centered on the elucidation of ribosomal components involved in peptidyl transfer. Attempts at unraveling components involved in the second class of interactions are still very few.
[73]
RIBOSOMAL FUNCTIONAL SITES
627
M a p p i n g o] C o m p o n e n t s o] the P e p t i d y l t r a n s ] e r a s e C e n t e r R e a g e n t s . C o m p o u n d s used for affinity labeling are listed in T a b l e II. 2°-37 Probes are for the most p a r t derivatives of P h e - t R N A and in some cases of M e t - t R N A ~ ~t. F o r charging with phenylalanine, u n f r a c t i o n a t e d or purified t R N A from E. coli or y e a s t was used. A systematic comparison of affinity probes m a d e with the different t R N A s 22 did not reveal differences in labeling pattern. Analogs of the initiator t R N A were inv a r i a b l y prepared s t a r t i n g f r o m purified t R N A ~ ~t. M o d i f y i n g groups fall into two general categories: (1) a - g are chemically reactive (haloacetyl, p - n i t r o p h e n y l c a r b a m y l , c h l o r a m b u c y l ) ; (2) h - n are photosensitive ( e t h y l d i a z o m a l o n y l , aryl azide, aryl keto). C h e m ically reactive and photosensitive probes differ m a i n l y in two respects. C o m p o u n d s of the first t y p e can react only with nucleophilic groups on the ribosome, whereas the extensive chemical reactivity of the photolysis
19G. StSffler in "Ribosomes" (M. Nomura, A. Tissi~res, and P. Lengyel, eds.), p. 615. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. M. Pellegrini, H. Oen, and C. R. Cantor, Proc. Natl. Acad. Sci. U~.A. 69, 837 (1972). sl H. Oen, M. Pellegrini, D. Eilat, and C. R. Cantor, Proc. Natl. Acad. Sci. U.S.A. 70, 2799 (1973). s2M. Pellegrini, H. Oen, D. Eilat, and C. R. Cantor, J. Mol. Biol. 88, 809 (1974). ss j. B. Breitmeyer and H. F. Noller, J. Mol. Biol. 101,297 (1976). s4M. Sopori, M. Pellegrini, P. Lengyel, and C. R. Cantor, Biochemistry 13, 5432 (1974). 23D. Eilat, M. Pellegrini, H. Oen, Y. Lapidot, and C. R. Cantor, J. Mol. Biol. 88, 831 (1974). s~M. Yukioka, T. Hatayama, and S. Morisawa, Biochim. Biophys. Acta 390, 192 (1975). s7A. P. Czernilofsky and E. Kuechler, Biochim. Biophys. Acta 272, 667 (1972). ~sA. P. Czernilofsky, E. E. Collatz, G. St5ffier, and E. Kuechler, Proc. Natl. Acad. Sci. U~.A. 71, 230 (1974). ~ R. Hauptmann, A. P. Czernilofsky, H. O. Voorma, G. StSffier, and E. Kuechler, Biochem. Biophys. Res. Commun. 56, 331 (1974). SOE. S. Bochkareva, V. G. Budker, A. S. Girshovich, D. G. Knorre, and N. M. Teplova, FEBS Lett. 19, 121 (1971). 31L. Bispink and H. Matthaei, FEBS Left. 37, 291 (1973). ~sA. S. Girshovich, E. S. Bochkareva, U. M. Kramarov, and Y. A. Ovchinnikov, FEBS Lett. 45, 213 (1974). 3~N. Hsiung, S. A. Reines, and C. R. Cantor, J. Mol. Biol. 88, 841 (1974). ~4N. Hsiung and C. R. Cantor, Nucleic Acids Res. 1, 1753 (1974). ~ A. Barta, E. Kuechler, C. Branlant, J. Sriwidada, A. Krol, and J. P. Ebel, FEBS Lett. 56, 170 (1975). 3~N. Sonenberg, M. Wilchek, and A. Zamir, Proc. Natl. Acad. Sci. U.S.A. 72, 4332 (1975). ~ N. Sonenberg, M. Wilchek, and A. Zamir, Abstr. Int. Congr. Biochem. lOth, Hamburg, 03-3-160; and Biochem. Biophys. Res. Commun. 72, 1534 (1976).
628
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
TABLE II PEPTIDYL-tRNA ANALOGS Aminoacyl-tRNA modified
N-blocking group
References
(a) Phe-tRNA
BrCI-I2C--
20-23
(b) M e t - t R N A ~ et
BrCI-I~C--
24
(c) Phe-tRNA
o II Br CI~C(GIy).--
25
(d) Phe-tRNA
Ic~c--
(e) Phe-tRNA
O~N~--
O I[
O II
26 o
O-- ~--
27, 28
0 29
(f) Met-tRNAf~*t
?, (g) Phe-tRNA
(C1CH,CH~)¢---N
30
(CH2)s-- C - O
II
/C - -
(h) Phe-tRNA
N,--C
31
C--OC2H s It o 0 (i) Phe-tRNA
Ns - ~
II C--
32
NO,
(j) Phe-tRNA
Ns
0 NO2
CH~---C--
33
[73]
RIBOSOMAL FUNCTIONAL SITES
629
TABLE II--Continued Aminoacyl-tRNA modified (k) Phe-tRNA
N-blocking group O N ___
RIBOSOMAL FUNCTION&L SITES
621
[73] Affinity Labeling of Ribosomal Functional Sites By ADA ZAMIR Ribosomes perform multiple functions in the course of protein synthesis; recognition and binding of the initiation region of mRNA, decoding of mRNA by binding of cognate aminoacyl-tRNAs, catalysis of peptide bond formation, and the translocation of nascent peptidyl-tRNA and mRNA. In addition, ribosomes can specifically bind a number of antibiotic compounds that interfere with one or another facet of protein biosynthesis. This diversity of function is matched by the complexity of ribosomal structure, an intricate arrangement of protein and RNA molecules, whose highly cooperative interaction is responsible for the formation of ribosomal functional sites. 1 This unique feature of the ribosome greatly obstructs the identification of ribosomal components directly located at specific active centers, and it renders difficult the distinction between directly and indirectly involved components. One approach that in principle circumvents this difficulty is based on the method of affinity labeling. Employing to date several classes of chemically or photoreactive site-specific ligands, this method has been applied to identify ribosomal components located within close range of several functional sites on the Escherichia coli ribosome. This chapter reviews methods applied in affinity labeling studies of ribosomes and describes the sites under study, major types of ligand used, criteria for establishing the specificity of labeling, and the characterization of modified ribosomal components. It does not include procedures for the synthesis of affinity labeling probes or a detailed discussion of the results obtained in the different studies. These can be found in the cited publications or elsewhere in this volume. For a general consideration of particular problems related to affinity labeling of ribosomes, see Cantor et al? and this volume [][5]. Early experiments are reviewed by Pongs et al2
mRNA Analogs Site Studied. In the normal functioning of the ribosome, mRNA initially binds to the ribosome in a manner allowing translation to start 1 H. G. Wittmann, Eur. d. Biochem. 61, 1 (1976). 2 C. R. Cantor, M. Pellegrini, and H. Oen, in "Ribosomes" (M. Nomura, A. Tissi~res, and P. Lengyel, eds.), p. 573. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. See also this volume [15]. 8O. Pongs, K. H. Nierhaus, V. A. Erdmann, and H. G. Wittmann, FEBS Lett. 40 (suppl.), 28 (1974).
622
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
at the correct initiation codon. Initiation and subsequent codons will then be decoded by selection and binding to the ribosome of the respective cognate aminoacyl-tRNAs. This process is actively affected by the ribosome. 4 Affinity labeling studies of the decoding site (defined as the ribosomal site in contact with the codon to be decoded) are based on the existence of relatively simple model compounds for mRNA. These include short oligonueleotides regarded as single isolated codons, or homopolynueleotides such as poly(U). Comparable to codons in natural mRNA, such analogs can direct binding of cognate aminoacyl-tRNAs to the ribosome and can, therefore, be considered to bind at the ribosomal decoding site. Two such sites may exist on the ribosome corresponding to the donor and acceptor positions of tRNA derivatives. While the initiation triplet is likely to direct binding to the donor site, all other triplets are decoded at the acceptor site. Reagents. Table I specifies all mRNA analogs reported to date as affinity labeling probes for ribosomal decoding sites. T M The table lists a group of oligonucleotides of defined length and sequence where chemically reactive groups have been introduced as substituents of the phosphate (a-c), pyrimidine ring (d-f), or ribose (g) groupings. Whereas the haloacetyl group in compounds a-f will make likely a reaction with nucleophilic groups on ribosomal proteins, reagent g was designed to interact with G residues in the rRNA. In addition, the list includes poly (U) and poly (4-thio-U), both of which are photoreactable. Oligonucleotides, Tests o] Function
The binding and coding properties of modified oligonucleotides were examined in order to establish their adequacy as affinity labeling probes. ' L. Gorini, Nature (London), New Biol. 234, 261 (1971). 50. Pongs and E. Lanka, Proc. Natl. Acad. Sci. U~S.A. 72, 1505 (1975). 60. Pongs, G. StSffier,and E. Lanka, J. Mol. Biol. 99, 301 (1975). O. Pongs and E. Rossner, Nucleic Acids Res. 3, 1625 (1976). s j. Petre and D. Elson (1976). Submitted for publication. "R. Liihrmann,U. Schwarz, and H. G. Gassen,FEB8 Lett. 32, 55 (1973). x0R. Lfihrmann,H. G. Gassen, and G. StSffier,Eur. J. Biochem. 66, 1 (1976). n O. Pongs, G. StSffler,and R. W. Bald, Nucleic Acids Res. 3, 1635 (197~). n R. Wagner and H. G. Gassen, Biochem. Biophys. Res. Cammun. ~ $19 (1975). 11I. Fiser, K. It. Seheit, G. StSffier, and E. Kueehler; /~o~'hem. ~ y s . Res. Commun. 601 1112 (1974). 1~E. ~mehl~r,. A. Ba~ta, L Fiser,, tL i~au~ma~m,, F. l~argaritella, W. Maurer, and G. ~6]~er;,A b s ~ In~. C~ngr. B~och~m. lOth, l~,ambur~03-3-153 (1976). 15M. L. Schenkman, D. C: Wards and P. B. Moore, Biochim. Biophys. Acta 353, 503 (1974). I. Fiser, P. Margaritella, and E. Kuechler,FEBS Left. 521 281 (1975).
[73]
623
RIBOSOMAL FUNCTIONAL SITES
TABLE I mRNA ANALOGS Reagent"
Modified group
?, (a) p*ApUpG
(b) p*UpGpA
(c) p*ApApA
0~~NH--
References
?,
0 tl HO--~--O-~ O~NH--C--CH~Br
O tl HO--P--O~--. I
/f-'~\
HN-~~Ntt-O
6
C--CH~Br 7
O
II
C-- CI~ Br
8
O
(d) UpUpUpU*
l O
O
(e) GpUpUpU*
I O (f) ApUpGpU*
O
O~N.!
11
I O
(g) UpUpUpU**
CH~O--
12
OO HN OH tt H /~----h J H--C--C~C----O (h) Poly(4-thio-U)
13, 14
(i) Poly(U)
15, 16
a Asterisks indicate modified component.
624
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
Although these tests are best performed in the absence of covalent binding, in most cases no care has been taken to avoid irreversible binding during the assay. Thus, for example, GpUpUpU ~ has been shown to direct the binding of Val-tRNA to an even greater extent than the nonmodified oligonucleotide,TM possibly reflecting a stabilization due to the irreversible binding of template. However, other oligonucleotide probes did not exhibit such enhanced activity, e'g" 11 Another criterion for functionality of short oligonucleotide templates is the stimulation of their binding to ribosomes by cognate aminoacyltRNAs. 8 Labeling Reaction. Labeling media were buffered at pH 7.2 to 7.4 and usually contained: 6-20 mM Mg 2÷, 80-150 mM NH4C1, and radioactively labeled affinity reagent at 10- to 100-fold excess over ribosomes added in the 70 S form or as isolated 30 S subunits (sometimes preactivated17). To enhance the specificity and stability of the reversible complex of oligonucleotide and ribosome, some reaction mixtures were supplemented with the cognate aminoacyl-tRNAs. 8,~°,12 Labeling mixtures were usually incubated for 1-2 hr at 0°-37% Maximal labeling of 30 S subunits or 70 S ribosomes by the various reagents ranged between 0.1 and 0.7 mole of reagent per mole of ribosome. However, the routine determination of reagent uptake by Millipore filtration does not appear to be reliable, since the values measured by this method by far exceed the recovery of label after partial fractionation2 The efficiency and pattern of labeling are markedly affected by the state of the ribosomes. Differences in reaction pattern were noted with 70 S ribosomes as compared to 30 S subunits, 6 with freshly prepared and stored 70 S ribosomes, 6 and with crude and salt-washed 70 S ribosomes or 30 S subunits. 1~ Particularly striking are the changes observed in the labeling of 30 S subunits with p~ApUpG, where freezing and thawing of the reaction mixture greatly enhanced uptake by making available new labeling sites on the ribosome2 Specificity o] Labeling. Several tests have been performed to determine whether affinity labeling probes were attached at functionally significant sites on the ribosome. The most crucial examination of the specificity of labeling is based on the ability of the modified ribosomes to bind the tRNA specified by the covalently bound oligonucleotide in the absence of any free template. Such tests were performed, for example, with p~ApUpG-modified 30 S subunits where IF-2 stimulated binding of fMet-tRNA~ et was demon1TA, Zamir, R. Miskin, and D. Elson, J. Mol. Biol. 60, 347 (1971).
[73]
RIBOSOMAL FUNCTIONAL SITES
625
strafed, 6 and with GpUpUpU~-modified 70 S ribosomes that were shown to be capable of binding Val-tRNA. 1° These results indicate that at least part of the modification in these cases has occurred at genuine decoding sites. Another criterion for specificity is based on the stimulation of covalent binding by cognate aminoacyl-tRNAs, s,l~ Reduced covalent reaction to ribosomes precomplexed with poly(U) and Phe-tRNA 1° and competition by nonmodified oligonucleotides 5,8 have also been taken as indications for the specificity of labeling. As indicated above, it is possible that codons bind to ribosomes at two positions corresponding to donor and acceptor sites. The nature of the site occupied by attached oligonucleotides has only been tentatively determined by testing the reactivity in the puromycin reaction of the aminoacyl-tRNA bound to the modified ribosomes2 ,1° In another case, the ability of ApUpGpU~-modified 70 S ribosomes to bind Met-tRNA~ et in the presence of EF-Tu has been considered indicative of acceptor site location of the bound codon. ~1
Polynucleotides Very little use has been made so far of polynucleotides as affinity labeling probes. The only reported cases are poly (U)15,16 and its analog poly (4-thio-U).13,14 Both can be made to react with ribosomes by irradiation at 253 nm and 335 nm, respectively. The adequacy of poly (4-thio-U) to serve as probe was indicated by its ability to direct Phe-polymerization. TM In both cases irradiated mixtures contained Phe-tRNA in addition to 70 S ribosomes and polynucleotide. As cross-linking of mRNA to the ribosome will probably arrest translocation, loss of polymerizing activity has been taken to indicate the specificity of binding. 18 It is worth noting that the average chain length of the polynucleotide probe is likely to affect the course of the reaction, as the use of long poly(U) chains has been reported to result in the formation of large cross-linked ribonucleoprotein aggregates. ~6 Analysis o] Modified Ribosomal Components. As a general rule in affinity labeling of ribosomes, excess of reagent was removed from the labeled ribosomes before any fractionation was attempted, to avoid any reaction from taking place after the structure of the ribosome had been disrupted. In cases in which 70 S ribosomes were labeled, the first step was normally the dissociation into subunits and determination of label u W. B~hr, P. Faerber, and K. H. Scheit, Eur. J. Biochem. 33, 535 (1973).
626
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
[73]
distribution in the two subunits, and subsequently in separated protein and RNA fractions. All mRNA analogs listed in Table I modified specifically the 30 S subunit, and all but compound g, which modified 16 S RNA, reacted with proteins. Modification with oligonucleotides introduces some difficulty in the identification of the labeled proteins. The attachment of a highly charged oligonucleotide probe or polynucleotide fragment alters considerably the net charge of the labeled protein and hence its electrophoretic mobility. This makes unreliable the identification of proteins by their position on the map obtained by 2-dimensional gel electrophoresis. ~,s This problem has been considered in the design of compound c, where, after the labeling reaction, the bulk of the oligonucleotide can be removed by a mild acid hydrolysis of the phosphoamide linkage2 In the other cases, partial splitting of proteins from 30 S subunits by cesium chloride gradient centrifugation6 and application of various one-dimensional gel electrophoretie techniques (quoted in references cited in footnotes 6, 10, 11) allowed a characterization of modified proteins. The most reliable and sensitive techniques for identifying the oligonucleotide-labeled proteins employ antibodies raised against purified individual ribosomal proteins. 19 Techniques used were radioimmunodiffusion, 6,1° and sucrose gradient centrifugation of soluble antigen-antibody complexesJ 8
Peptidyl-tRNA and tRNA Derivatives Sites Studied. Interactions of tRNA with the ribosome can be broadly classified into two types. (a) Interactions with the peptidyltransferase center of the 50 S ribosomal subunit involving the 3' terminus of peptidylor aminoacyl-tRNA occupying, respectively, the donor (peptidyl) or acceptor (aminoacyl) site of the catalytic center. (b) Interactions likely to involve internal parts of the tRNA molecule and designed to align and stabilize the binding of cognate tRNAs to the ribosome. Affinity labeling probes for mapping of ribosomal components involved in interactions of the first type were constructed by introducing suitable substituents at the free terminal amino group of aminoacyl or peptidyl tRNA. To study the second class interactions, sites within the tRNA molecule must be rendered capable of reacting with potential binding sites on the ribosome. Most studies to date have centered on the elucidation of ribosomal components involved in peptidyl transfer. Attempts at unraveling components involved in the second class of interactions are still very few.
[73]
RIBOSOMAL FUNCTIONAL SITES
627
M a p p i n g o] C o m p o n e n t s o] the P e p t i d y l t r a n s ] e r a s e C e n t e r R e a g e n t s . C o m p o u n d s used for affinity labeling are listed in T a b l e II. 2°-37 Probes are for the most p a r t derivatives of P h e - t R N A and in some cases of M e t - t R N A ~ ~t. F o r charging with phenylalanine, u n f r a c t i o n a t e d or purified t R N A from E. coli or y e a s t was used. A systematic comparison of affinity probes m a d e with the different t R N A s 22 did not reveal differences in labeling pattern. Analogs of the initiator t R N A were inv a r i a b l y prepared s t a r t i n g f r o m purified t R N A ~ ~t. M o d i f y i n g groups fall into two general categories: (1) a - g are chemically reactive (haloacetyl, p - n i t r o p h e n y l c a r b a m y l , c h l o r a m b u c y l ) ; (2) h - n are photosensitive ( e t h y l d i a z o m a l o n y l , aryl azide, aryl keto). C h e m ically reactive and photosensitive probes differ m a i n l y in two respects. C o m p o u n d s of the first t y p e can react only with nucleophilic groups on the ribosome, whereas the extensive chemical reactivity of the photolysis
19G. StSffler in "Ribosomes" (M. Nomura, A. Tissi~res, and P. Lengyel, eds.), p. 615. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1974. M. Pellegrini, H. Oen, and C. R. Cantor, Proc. Natl. Acad. Sci. U~.A. 69, 837 (1972). sl H. Oen, M. Pellegrini, D. Eilat, and C. R. Cantor, Proc. Natl. Acad. Sci. U.S.A. 70, 2799 (1973). s2M. Pellegrini, H. Oen, D. Eilat, and C. R. Cantor, J. Mol. Biol. 88, 809 (1974). ss j. B. Breitmeyer and H. F. Noller, J. Mol. Biol. 101,297 (1976). s4M. Sopori, M. Pellegrini, P. Lengyel, and C. R. Cantor, Biochemistry 13, 5432 (1974). 23D. Eilat, M. Pellegrini, H. Oen, Y. Lapidot, and C. R. Cantor, J. Mol. Biol. 88, 831 (1974). s~M. Yukioka, T. Hatayama, and S. Morisawa, Biochim. Biophys. Acta 390, 192 (1975). s7A. P. Czernilofsky and E. Kuechler, Biochim. Biophys. Acta 272, 667 (1972). ~sA. P. Czernilofsky, E. E. Collatz, G. St5ffier, and E. Kuechler, Proc. Natl. Acad. Sci. U~.A. 71, 230 (1974). ~ R. Hauptmann, A. P. Czernilofsky, H. O. Voorma, G. StSffier, and E. Kuechler, Biochem. Biophys. Res. Commun. 56, 331 (1974). SOE. S. Bochkareva, V. G. Budker, A. S. Girshovich, D. G. Knorre, and N. M. Teplova, FEBS Lett. 19, 121 (1971). 31L. Bispink and H. Matthaei, FEBS Left. 37, 291 (1973). ~sA. S. Girshovich, E. S. Bochkareva, U. M. Kramarov, and Y. A. Ovchinnikov, FEBS Lett. 45, 213 (1974). 3~N. Hsiung, S. A. Reines, and C. R. Cantor, J. Mol. Biol. 88, 841 (1974). ~4N. Hsiung and C. R. Cantor, Nucleic Acids Res. 1, 1753 (1974). ~ A. Barta, E. Kuechler, C. Branlant, J. Sriwidada, A. Krol, and J. P. Ebel, FEBS Lett. 56, 170 (1975). 3~N. Sonenberg, M. Wilchek, and A. Zamir, Proc. Natl. Acad. Sci. U.S.A. 72, 4332 (1975). ~ N. Sonenberg, M. Wilchek, and A. Zamir, Abstr. Int. Congr. Biochem. lOth, Hamburg, 03-3-160; and Biochem. Biophys. Res. Commun. 72, 1534 (1976).
628
NUCLEIC ACIDS AND RIBOSOMAL SYSTEMS
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TABLE II PEPTIDYL-tRNA ANALOGS Aminoacyl-tRNA modified
N-blocking group
References
(a) Phe-tRNA
BrCI-I2C--
20-23
(b) M e t - t R N A ~ et
BrCI-I~C--
24
(c) Phe-tRNA
o II Br CI~C(GIy).--
25
(d) Phe-tRNA
Ic~c--
(e) Phe-tRNA
O~N~--
O I[
O II
26 o
O-- ~--
27, 28
0 29
(f) Met-tRNAf~*t
?, (g) Phe-tRNA
(C1CH,CH~)¢---N
30
(CH2)s-- C - O
II
/C - -
(h) Phe-tRNA
N,--C
31
C--OC2H s It o 0 (i) Phe-tRNA
Ns - ~
II C--
32
NO,
(j) Phe-tRNA
Ns
0 NO2
CH~---C--
33
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RIBOSOMAL FUNCTIONAL SITES
629
TABLE II--Continued Aminoacyl-tRNA modified (k) Phe-tRNA
N-blocking group O N ___
