Biochimie (199 l) 73, 639-645 © Soci6t6 frangaise de biochimie et biologie mol6culaire/ Elsevier, Paris
639
Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties ER Dabbs Department of Genetics, University of the Witwatersrand, Johannesburg. PO Wits 2050, South Afi'ica
(Received 8 November 1990; accepted i 2 March 1991)
Summary - - We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli
ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacteriai ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle. ribosome / ribosomal mutants / ribosomal protein
Introduction
The Escherichia coli ribosome has a complement of about 55 different proteins [1]. There is a very high degree of correspondence between these proteins and those of a representative Gram-positive bacterium, Bacillus subtilis [2]. The conservation of the protein complement over long evolutionary time suggests that each ribosomal protein is important in some aspect of organelle structure, function, regulation, or assembly. However, notwithstanding a large amount of early work, the role of many proteins in ribosome function or regulation remained obscure. The primary sequence of every E coli ribosomal protein has been established [3]. Most progress has been made in the fields of organelle topography and organelle assembly. Already in 1974, most small subunit proteins had been assigned a position in the assembly map [4] and by 1982 an assembly map had likewise been drawn up for the large subunit [5]. Initial approaches to understanding the function of each protein were made using single protein omission experiments [4] but this required the separation of all r-proteins and then reconstitution of ribosomal RNA with all proteins except one. Obviously, a much simpler means of achieving the same ends, and one that avoids complications such as protein denaturation and renaturation, would be to obtain mutants lacking individual ribosomal proteins.
Several selection systems in E coli generated mutants lacking small subunit protein $20 [6-8], but no other proteins were ever found to be missing. In two Bacillus species, on the other hand, mutants with large subunit protein L ll missing were identified [9, 10]. The same selection was used for both species, ability to grow in the presence of the antibiotic thiostrepton. However, this se',~:ction could not be applied to E coli since Gram-negative bacterial cell walls are impermeable to thiostrepton and hence these organisms are resistant to the antibiotic. To develop a selection that produced diverse proteins absent from the ribosome, the starting point was the work of Gorini [ l 1]. They used streptomycindependent mutants to isolate spontaneous second site suppressor mutations which abolished the antibiotic requirement. The suppressor mutations possessed on their own a ribosomal ambiguity (ram) phenotype. Subsequently, a modification of this selection permitted the isolation of suppressor mutations which had a restrictive phenotype [ 12]. In both cases, the suppressor mutations mapped to only one or two loci and the protein product of these genes was altered, not absent. Another streptomycin-dependent mutant was isolated which had the unique phenotype of generating spontaneous antibiotic-independent revertants with alterations in any of a large number of ribosomal proteins [13]. An exhaustive analysis of these revertants revealed
640
ER Dabbs
that mutational alternations could be obtained for every single ribosomal protein. This indicated that earlier explanations of the reason why mutational alterations were identified in only a few ribosomal proteins, vi2 that lesions in the rest of the proteins could be lethal, were invalid. To broaden the approach to obtain spontaneous ribosomal protein mutants, other antibiotics were substituted for streptomycin. A large number of antibiotics have been shown to have the ribosome as their target moiety, and detailed investigation has generally shown that these agents act at a number of different points, both in terms of the ribosomal function they inhibit and in terms of the binding site on the organelle [14-18]. It is likely therefore that mutants dependent on different antibiotics will possess lesions with different properties, and that suppressor mutations for each class of dependent strain may differ.
sistent with this, the phenotype of the latter is found to be the product of two mutations in every case investigated in detail [ 19-21 ], one being a mutation conferring antibiotic resistance, and the second converting the resistance to dependence. This contrasts with the single mutations responsible for the streptomycin dependent phenotype. Mutants dependent on spectinomycin [ 19], erythromycin [22], or kasugamycin [21] were obtained. Analysis of revertants obtained from these revealed that the second of these three was the most productive in generating strains with a spectrum of altered ribosomal proteins [22]. Moreover, in many cases, revertants had one or two proteins missing. Several erythromycin dependent mutants were isolated and each generated ribosomal protein (r-protein) lacking mutants. Some kasugamycin dependent mutants exhibited the same property, while the one spectinomycin strain gave rise to mutants lacking large subunit protein L 1 [ 19].
Materials and methods
Identification of mutants
These were generally as described in [6, 12, 13]. Other methods are described in the articles cited. Results Whereas spontaneous streptomycin-dependent mutants may readily be obtained, strains requiring other antibiotics for growth only arise after mutagenesis. ConTable I. Ribosomal proteins missing in mutants of E coil
Protein
Mutant designation
LI LI I Li5 LI9 L24 L27 L28 L29 L30 L33 SI $6 $9 S13 S 17 $20
RDI9, MVI7-10 AM68, AM76, AM77 AMI6-98 AMI49 AM290 AMI25 AM81, AM 108 AM111 AMI0 AM90, AM 108 VTS03 AM80 AM83 AM109-113 AM 111 VT514
Pheno~pe Reference cs ts cs cs cs cs ts ts
[26] [27] [28] [28] [29] [22] [22] [221 [22] [22] [251 unpublished [22] unpublished [22] [25]
Ribosomal proteins were initially resolved on twodimensional polyacrylamide gels [23], stained with amido-black. A protein spot that was apparently missing in the gel of a mutant may simply mean that it had altered mobility and co-migrated with another protein. To determine that such was not the case, a battery of immunological tests were performed. After initial screening with Ouchterlony double diffusion gels, additional tests were made using a modified immuno-electrophoresis technique on cellulose acetate gels. This had the advantage that antibodyantigen complexes did not have to give rise to an insoluble precipitate for cross-reacting material to be detected. Subsequently, immunoblotting has been used; material resolved in SDS slab gels was blotted onto nitrocellulose filters and these were treated with antiserum before immuno-peroxidase staining [24].
Ribosomal proteins missing in mutants of E coli As shown in table I, mutants with any one of sixteen of the fifty-five ribosomal proteins have been found. In every case except that of protein S1 the initial identification was on the basis of two-dimensional polyacrylamide gel electrophoresis (2--D PAGE), with subsequent confirmation by immunological methods. Again with the single exception of protein S 1, protein cross-reacting material (crm) undetectable on the ribosome was also not detected in extracts from whole cells. Therefore, it was not simply a case of the protein being more easily washed off the ribosome. The S1 mutant was found using solely immunological methods, and whereas there was no crm on the ribosome there was crm in whole cell extracts.
Ribosomal protein lacking mutants
Nature of the mutation in r-protein-lacking strains Genetic analysis of r-protein-lacking mutants revealed that in all mutants except S1, the lesion responsible was close to and probably within the gene for the protein which was missing. Apart from protein L24, all proteins found to be lacking have genes either near the end of large transcription units (eg S17, L15, L29, L30) or else, genes that are part of small transcription units (eg $9, $20, L1, L11, L19, L30) [30]. Southern blots from a number of mutants indicated that the DNA fragment bearing the relevant gene was not smaller in the mutant than in a wild-type (E Dabbs and N Fiil, unpublished). Therefore, it was improbable that deletions were responsible for the observed loss, although it could not be excluded that small deletions had occurred which changed the phase of the reading frame. Where there was a size change in the DNA fragment, it was larger than in wild-type. Some of the mutant phenotypes may be due to insertion sequences, as has been found for certain other ribosomal mutants [311. In one case, the nature of the lesion causing an rprotein to be lacking has been determined by. DNA sequencing this being an rplX mutation causing the loss of prot,:in L24 [32]. An AAA codon had changed to a TAA codon at position 61 in the gene, so instead of a lysine codon there was a stop codon [33]. As a result of the base substitution, a 20 amino-acid peptide should be generated instead of the 104 residue wildtype protein L24. A 20 amino-acid peptide should be detectable with the immunological techniques employed. The fact that it was not would suggest that the fragment is rapidly degraded. The rplX lesion conferred a temperature-sensitive conditional lethal phenotype on the cell, which permitted the selection of temperature-resistant revertants. DNA from three revertants was analysed and it was found that the TAA codon had mutated to GAA, TCA, or TTA [33]. Therefore, instead of an ochre stop codon a glutamic acid, serine or leucine residue would be present. Consistent with this, 2-D PAGE of r-proteins from these revertants revealed each possessed a full size protein L24 but of a form electrophoretically distinct from wild-type.
Phenotype arising from r-protein-lacking mutations Generally the lesions responsible for a protein being missing did have an effect on phenotype, as was seen when P1 transduction was used to transfer the mutation out of the strain in which it was isolated. This reduced the likelihood that phenotypes were due to other mutations arising from the mutagenesis employed before selection of the original dependent strain.
641
In all mutants except those lacking L29 or L30, the generation time was significantly increased. Additionally, there was sometimes either a temperature-sensitive (S17, $20, L24) or a cold-sensitive ($9, L15, L27, L28, L33) conditional lethal phenotype. Selection for reversion of the conditional lethal phenotype resulted in strains in which the missing protein was frequently restored: this was investigated for the S 17, L15, L24, L27 and L28 mutants and found to be the case (E Dabbs, unpublished). Such findings were further evidence that the original lesions responsible were not deletions in the genes for these proteins.
Lacking mutants as tools to investigate the ribosome Mutants with individual ribosomal proteins missing offer a simple tool to investigate the role of these proteins. Ribosomes of these strains provide a ready source of intact ribosomes with all proteins except one present at normal stoichiometry. They avoid the need to employ laborious fractionation and reconstitution procedures to arrive at the same end. These mutants have been used as a tool in investigating ribosome topography, function and assembly.
Ribosome topography As stated earlier, all r-protein-lacking mutants have been studied using immt~nological techniques. Many have then been used in experiments to determine the position of individual proteins on the ribosomal surface. Early work on ribosome topography had in some cases led to misidentification of antigenic determinants, which may have reflected non-specific antibody binding or else the presence in purified protein fractions used to raise antibodies of trace amounts of highly immunogenic contaminating material. To avoid misidentification of epitopes on the subunit surface, mutants with individual proteins missing provide an ideal control. This is because any binding sites present in both wild-type and mutant must be due to non-specific binding, whereas sites seen in electron micrographs of subunits from the wild-type but not the mutant very probably are true sites for the protein in question. The location of a protein in particles from wild-type and from a mutant in which the protein missing had been added back was invariably indistinguishable. It was found that the missing protein was rapidly and stoichiometrically incorporated into subunits from the mutant when it was added to a suspension of these particles. This reconstitution occurred under a wide range of conditions, with no special procedure or buffer being necessary. Mutants which have been employed as part of the topographical pinpointing have included one in which proteins L l, L I 1, L I5, LI9 or S17 were missing [34, 35].
642
ER Dabbs
Functional changes in mutant strabts Mutants lacking a particular-protein are an ideal tool to explore the function of that protein. Except for the missing protein, all others are present in the ribosome at the same stoichiometry as in wild-type. The selection which generated strains deficient in a particular protein was spontaneous, which minimises the risk that the altered properties are due to additional lesions not detectable on the basis of changes in 2-D gel analysis. Furthermore, since the missing protein can be readily restored to ribosomal particles, then by titrating back the protein and at the same time monitoring ribosomal properties one can confirm that the altered property is result of the absence of that protein. Mutants lacking protein L I were the first to be studied for changes in organelle function. Ribosomes from such mutants had only about half the in vitro polypeptide synthetic activity of wild-type, but this could be restored to wild-type levels by the addition of purified protein L I to the ribosome suspension prior to assaying activity [36]. In subsequent experiments, it was shown that both the binding of N-acetylPhe-tRNA to ribosomes and the 6-8 fold stimulation of the elongation factor G (EF-G)-dependent GTPase reaction by mRNA together with tRNA were greatly reduced when large subunits from L I mutants were used [37]. A supplement of purified protein L1 again restored these activities to wild-type levels. The basic non-mRNA and non-tRNA dependent EF-G GTPase was not diminished in assays using the mutant ribosomes. This work led to the conclusion that protein L 1 is involved in the interaction between ribosomes and peptidyl tRNA in the peptidyl site [37]. Mutants lacking protein L II have been used to explore the binding to the ribosome of factors in translation. Specifically, ribosomal particles from these strains have been found to have changes in the binding of release factors 1 and 2 [38]; addition of protein L I 1 to mutant ribosomes again resulted in a return to the wild-type pattern of release factor binding. This was consistent with reconstitution experiments that were used to identity ribosomal components involved in factor binding [39]. These L I 1 lacking strains have also been investigated in terms of the subunit interaction. It was found that the mechanism of association of mutant 50S with 30S subunits, or the structure of the resulting 30S-50S couples, is altered in such a way as to cause ejection of an AcPhe-tRNA molecule prebound to the 30S subunits in response to polyU [40]. 30S ribosomal subunits from a strain in which protein $20 was missing were found to be defective in two respects [41]. They have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits. Also, they are defective in functions which
do not require their interaction with the large subunit, such as the formation of tertiary complexes with aminoacyl-tRNAs and templates, including the formation of 30S initiation complexes with f Met-tRNA and mRNA. Experiments on other mutants are in progress. In some cases, proteins about which very little is known beyond their sequences, such as L28 and L33, have a much more drastic effect when they are absent than a protein which has been ascribed an important role in the ribosome (LI5). Strains lacking L28 or L33 should help clarify what the part is these proteins play in the ribosome. Subunit assembly in r-protein mutants Most of the proteins for which mutants missing them have been isolated are not ones which are involved in early stages of subunit assembly [4, 5], This is in agreement with the observation that most of these mutants show no abnormalities or defects in assembly. However, two large subunit proteins which have been attributed important roles in organelle assembly, L I5 and L24, are amongst those proteins for which mutants lacking them have been isolated. Protein L24 is, together with L3, one of the two initiator-proteins which first binds to the large subunit [42]. L24 is essential for early large subunit assembly, but is dispensable in later steps and for organelle function [43]. Strains carrying the rplX mutation which leads to absence of protein L24 have a much reduced growth rate, about six-fold slower than wild-type at permissive temperatures. The subunit profile at 30°C reveals a considerably diminished large subunit peak [32] which moreover migrates at 47S rather than 50S [29]. The strain is also temperature-sensitive. In the standard two-step reconstitution, large subunits from L24- mutant showed that - as expected - only one initiator-protein was effective. When the temperature of the first step was reduced from 44 to 26°C however, active particles were reconstituted at about 50% efficiency even though L24 was not present. With this change in conditions, there were again two initiatorproteins. This meant that an additional protein, identified as L20 [44] was present which could replace L24 as initiator of assembly at the lower temperature. This in vitro situation parallels the in vivo situation, in that the L24 lacking mutant can grow at the low end of the normal E coli range of growth temperature. In contrast, strains containing the rplO mutation leading to the absence of protein L15 grow well and ribosomal subunit profiles are similar to that of wildtype, yet protein L 15 has been attributed a pivotal role
Ribosomal protein lacking mutants
in 50S assembly [5]. The homologue of protein L15 in a Bacillus strain does not occupy the same important position in assembly and it may be that E coli possesses alternative pathways which circumvent the requirement for-protein L15 [45].
Changes in gene expression There are at least twenty-five transcriptional units in E coli which give rise to products involved in the translation machinery of the cell [46]. Notwithstanding this plethora, there is coordination in their expression such that balanced synthesis of the components occurs. Mutants lacking individual r-proteins have been used to investigate regulation of this expression. Immunological [27] and pulse labelling [47] studies have been made on mutants lacking protein L1. Both experiments have shown that these strains have elevated levels of protein L11 but not any other ribosomal protein. L11 mutants, on the other hand, have levels of protein L I which are if anything less than normal [27]. The data are fully consistent with in vivo studies which have indicated that the expression of the L l l - L I transcriptional units is regulated by protein LI but not by protein LI 1" when protein L1 is absent from a cell, then increased synthesis rates and increased intracellular levels of protein L 11 would be anticipated.
Ribosomal protein homologies in different organisms Mutant ribosomes can readily incorporate the missing protein when this is added to a suspension of the particles. This incorporation occurs irrespective of whether purified protein or a mixture of ribosomal proteins is added. Moreover, it is found that there is incorporation even if the total ribosomal protein (TP) of an organism other than E coli is used. If there is prior incubation with excess TP from E coli, however, then incorporation of heterologous proteins does not Occur.
These observations have been used as a method of identifying ribosomal correspondences between evolutionarily remote organisms. It has the virtue that no prior fractionation and purification of individual proteins and sequencing or immunology is necessary. This method has been used to identify the B subtilis protein which corresponds to E coil protein L25 [48], and the archaebacterium Methanococcus vannielii protein which corresponds to E coli protein L1 (G St~ftier, personnal communication).
Why do r-protein-lacking mutants arise? As stated earlier, these mutants are generated in several selections. Mutants dependent on different antibiotics in some cases give rise to revertants with lesions in the same r-protein gene. Thus $20- mutants are generated as suppressors of both streptomycin and
643
erythromycin dependence [8, 22]. L l- strains occur amongst the revertants of erythromycin, spectinomycin, or kasugamycin dependents [19, 21, 22]. There are differences, since only erythromycin mutants generate strains missing any of the 16 proteins (table I). However, the parallels between the different selections suggest there must be elements in common. As an approach to understanding what this might be, a comparison was made of two mutants which had the same dependent phenotype but differed in that one generated revertants which included strains with proteins missing from the ribosome and one which did not [49]. Both parental strains were dependent on kasugamycin for growth. The results of experiments in which the two strains were deprived of antibiotics are shown in figure 1. For both, antibiotic removal was bacteriocidal. They both differed therefore from streptomycin dependent strains, where antibiotic removal was bacteriostatic. The two differed from each other in initial response to antibiotic removal: one mutant reacted to this by a surge in growth above that of cells maintained in the presence of kasugamycin. This was paralleled by a surge in protein synthesis [49]. The other mutant showed no such surge. The former strain was one which could be used to select r-protein-lacking mutants, whereas the latter could not. The data showed that the former mutant was dependent on kasugamycin because this antibiotic was needed to depress the level of protein synthesis. Removal led to enhanced protein synthesis, but this enhancement presumably resulted in a lethal disequilibrium within the cell: this was manifested in a cessation of cell growth and in cell death. Therefore, what sort of mutational event could rescue such a strain from the bacteriocidal effect of antibiotic removal? One class of events would be ribosomal mutations which slow dowr, the function of this organelle. This is in agreement with the observation that most lesions leading to an r-protein being missing cause a distinct slowing of growth. Presumably the main way in which ribosome function may be impaired is by loss of one of its consistuent proteins.
Other aspects Two additional uses to which r-protein-lacking mutants can be put are to clone r-protein genes and for crystallographic studies of the ribosome. As stated above, many of the lesions in these mutants generate a conditional lethal phenotype. Therefore, one might be able to clone the corresponding gene based on complementation of this phenotype. The cloning of rplX from E coli wild-type and pseudorevertant strains was done on this basis since the rplX lesion resulting in the absence of protein L24 conferred temperature sensitivity [33].
644
ER Dabbs Mutants with a protein missing from the ribosome can be used to facilitate the incorporation of this protein, to which a metal cluster has been attached (A Yonath, personal communication). Different heavy metal derivatives will be used to permit solution of Xray diffraction patterns, and hence aid in determining the structure of ribosomal particles.
A
Implications o f r-protein-lacking m u t a n t s
0'01t ,
I
,
,
0
2
4
6
TIME(HR)
B
1.0
Over one quarter of the E coli r-proteins have been found to be missing in at least one mutant. There is no reason to suppose that the proteins given in table I represent an exhaustive list of those proteins which are not essential because as long as additional revertants from the appropriate selections were analysed, additions were made to the list. Notwithstanding this, it seems likely that a subset of proteins is essential. One evidence of this may be reflected in the distribution of chloroplast r-protein genes between those that are nuclear encoded and those that are encoded by the chloroplast [50]. There is a strong correlation between the proteins identified as lacking and the chloroplast homologues that are nuclear encoded. Perhaps the set of essential proteins are the ones still encoded by the chloroplast chromosome, as well as of course being absent from table I.
References
o0 . ~ co C3 O
1
0.01
I
0
I,
2
I
t.
I
6
TIME (HR) Fig 1. Growth curves of strains MVIOI and PB67, with viable counts and OD 650 nm. A. MVI01 absorbance in presence ( o ) or absence ( • ) of kasugamycin; cell number in presence ((~) or absence (0) of antibiotic. B. PB67 absorbance in presence (D) or absence ( I ) of kasugamycin; cell number presence (A) or absence (&) of antibiotic. Arrows indicate time at which cells were transferred from antibiotic containing medium (50 l.tg/ml) to fresh medium. Based on data from [48].
1 Wittmann HG (1985) ln: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer (3. eds) SpringerVerlag. NY, 1-27 2 Higo K, Otaka E. Osawa S (1982) Mol Gen Genet 185. 239-246 3 Wittmann-Liebold B (1985) ln: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, NY, 326-361 4 Nomura M, Held WA (1974) ln: Ribosomes (Nomura M, "Tissi~re A, Lengyel P, eds) Cold Spring Harbor Laboratory, 193-223 5 R6hl R, Nierhans KH (1982) Proc Natl Acad Sci USA 79, 729-733 6 Pepersberg W, Geyi D, Hummel H, BOck A (1980) in: Genetics and Evolution of RNA Polymerase, tRNA, and Ribosomes (Osawa S, Ozeki H, Uchida H, Yura T, eds) University of Tokoy Press, 359-377 7 Isono S, Isono K, Hirota Y (1978) Mol Gen Genet 65, 15-20 8 Dabbs ER (1978) Mol Gen Genet 165, 73-78 9 Cundliffe E, Dixon P, Stark M, StUffier G, Ehrlich R, St6ffler-Meilicke M, Cannon M (1979) J Mol Biol 132, 235-252 10 Wienen B, Ehlrich R, St6ffler-Meilicke M, St6ffler G, Smith J, Weiss D, Vince R, Pestka S (1979) J Biol Chem 254, 8031-8041 11 Gorini L (1971) Nature New Bio1234, 261-264 12 Dabbs ER (1977) Mol Gen Genet 158, 55-61 13 Dabbs ER (1976) Mol Gen Genet 149, 303-309 14 Okuyama A, Tanaka N (1975) J Antibiotics 28, 903-905
Ribosomal protein lacking mutants 15 Wallace BJ, Tai PC, Davis BD (1973) J Mol Biol 75, 391--400 16 Wallace BJ, Tai PC, Davis BD (1974) Proc Natl Acad Sci USA 71, 1634-1638 17 Pestka S (1974) AntAgents Chemother 5, 255-267 18 Palmer E, Wilhelm JM (1978) Cell 13, 329-334 19 Dabbs ER (1977) Mol Gen Genet 151,261-267 20 Dabbs ER (1978)JBacteriol 136, 994-1001 21 Dabbs ER (1979)JBacteriol 139, 1072-1074 22 Dabbs ER (1979) J Bacteriol 140, 734-737 23 Kaltschmidt E, Wittmann HG (1970) Analyt Biochem 36, 401--412 24 St6ffler-Meilicke M, Dabbs ER, Albrecht-Ehrlich R, StSffler G (1985) Fur J Biochem 150, 485--490 25 Dabbs ER, Hasenbank R, Kastner B, Rak KM, Wartusch B, St6ffler G (1983) Mol Gen Genet 192, 301-308 26 Dabbs ER, Ehrlich R, Hasenbank R, Schroeter BH, StOffler-Meilicke M, St6ffler G (1981) J Mol Biol 149, 553-578 27 St6ffler G, Dabbs ER, Hasenbank R (1981) Mol Gen Genet 181,164-168 28 Lotti M, Dabbs ER, Hasenbank R, Stt~ffler-Meilicke M, St6ffler G (1983) Mol Gen Genet 192, 295-300 29 Herold M, Nowotny V, Dabbs ER, Nierhaus KH (1986) Mol Gen Genet 203, 281-287 30 Gourse RL, Sharroek RA, Nomura M (1985) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, NY, 766-788 31 Schnier J, lsono S, Cumberlidge G, lsono K (1985) Mol Gen Genet 199, 265-270 32 Dabbs ER (1982) Mol Gen Genet 187,453--458 33 Nishi K, Dabbs ER, Schnier J (1985) J Bacteriol 163, 890--894
645
34 St6ffler G, Cundliffe E, StOffler-Meilicke M, Dabbs ER (1980) J Biol Chem 255, 10517-10522 35 St~iffler G, Noah M, St6ffler-Meilicke M, Dabbs ER (1984) J Biol Chem 259, 4521--4526 36 Subramanian AR, Dabbs ER (1980) Fur J Biochem 112, 425--430 37 Sander G (1983) JBiol Chem 258, 10098-10103 38 'Fate WP, Dogni NJ, Noah M, St/Sffler G (1984) J Biol Chem 259, 7317-7324 39 Tate WP, Schulze H, Nierhaus KH (1983) J Biol Chem 258, 12816-12820 40 G6tz F, Fleisher C, Pon CL, Gualerzi CO (1989) Eur J Biochem 183, 19-24 41 G6tz F, Dabbs ER, Gualerzi C (1990) Biochim Biophys Act 1050, 93-97 42 Nowotny V, Nierhaus KH (1986) Proc Natl Acad Sci USA 79, 7238-7242 43 Spilimann S, Nierhaus KH (1978) J Biol Chem 253, 7047-7050 44 Franceschi FJ, Nierhaus KH (1988) Biochemistry 27, 7056--7059 45 Franceschi FJ, Nierhaus KH (1990) J Bioi Chem 265, 16676--I 6682 46 Nomura M, Gourse R, Baughman G (1984) Annu Rev Biochem 53, 75-117 47 Jinks-Robertson S, Nomura M (1981) J Bacteriol 145, 1445-1447 48 Dabbs ER (1983)JBacterioi 156, 966-969 49 Dabbs ER (1983)JBacteriol 153, 709-715 50 Subramanian AR, Smooker PM, Giese K (1990) In: The Ribosome, Structure, Function, and Evolution (Hill WE, Dahlberg A, Garrett RA, Moore PB, Warner JR, eds) American Society for Microbiology, Washington, 655--663
639
Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties ER Dabbs Department of Genetics, University of the Witwatersrand, Johannesburg. PO Wits 2050, South Afi'ica
(Received 8 November 1990; accepted i 2 March 1991)
Summary - - We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli
ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacteriai ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle. ribosome / ribosomal mutants / ribosomal protein
Introduction
The Escherichia coli ribosome has a complement of about 55 different proteins [1]. There is a very high degree of correspondence between these proteins and those of a representative Gram-positive bacterium, Bacillus subtilis [2]. The conservation of the protein complement over long evolutionary time suggests that each ribosomal protein is important in some aspect of organelle structure, function, regulation, or assembly. However, notwithstanding a large amount of early work, the role of many proteins in ribosome function or regulation remained obscure. The primary sequence of every E coli ribosomal protein has been established [3]. Most progress has been made in the fields of organelle topography and organelle assembly. Already in 1974, most small subunit proteins had been assigned a position in the assembly map [4] and by 1982 an assembly map had likewise been drawn up for the large subunit [5]. Initial approaches to understanding the function of each protein were made using single protein omission experiments [4] but this required the separation of all r-proteins and then reconstitution of ribosomal RNA with all proteins except one. Obviously, a much simpler means of achieving the same ends, and one that avoids complications such as protein denaturation and renaturation, would be to obtain mutants lacking individual ribosomal proteins.
Several selection systems in E coli generated mutants lacking small subunit protein $20 [6-8], but no other proteins were ever found to be missing. In two Bacillus species, on the other hand, mutants with large subunit protein L ll missing were identified [9, 10]. The same selection was used for both species, ability to grow in the presence of the antibiotic thiostrepton. However, this se',~:ction could not be applied to E coli since Gram-negative bacterial cell walls are impermeable to thiostrepton and hence these organisms are resistant to the antibiotic. To develop a selection that produced diverse proteins absent from the ribosome, the starting point was the work of Gorini [ l 1]. They used streptomycindependent mutants to isolate spontaneous second site suppressor mutations which abolished the antibiotic requirement. The suppressor mutations possessed on their own a ribosomal ambiguity (ram) phenotype. Subsequently, a modification of this selection permitted the isolation of suppressor mutations which had a restrictive phenotype [ 12]. In both cases, the suppressor mutations mapped to only one or two loci and the protein product of these genes was altered, not absent. Another streptomycin-dependent mutant was isolated which had the unique phenotype of generating spontaneous antibiotic-independent revertants with alterations in any of a large number of ribosomal proteins [13]. An exhaustive analysis of these revertants revealed
640
ER Dabbs
that mutational alternations could be obtained for every single ribosomal protein. This indicated that earlier explanations of the reason why mutational alterations were identified in only a few ribosomal proteins, vi2 that lesions in the rest of the proteins could be lethal, were invalid. To broaden the approach to obtain spontaneous ribosomal protein mutants, other antibiotics were substituted for streptomycin. A large number of antibiotics have been shown to have the ribosome as their target moiety, and detailed investigation has generally shown that these agents act at a number of different points, both in terms of the ribosomal function they inhibit and in terms of the binding site on the organelle [14-18]. It is likely therefore that mutants dependent on different antibiotics will possess lesions with different properties, and that suppressor mutations for each class of dependent strain may differ.
sistent with this, the phenotype of the latter is found to be the product of two mutations in every case investigated in detail [ 19-21 ], one being a mutation conferring antibiotic resistance, and the second converting the resistance to dependence. This contrasts with the single mutations responsible for the streptomycin dependent phenotype. Mutants dependent on spectinomycin [ 19], erythromycin [22], or kasugamycin [21] were obtained. Analysis of revertants obtained from these revealed that the second of these three was the most productive in generating strains with a spectrum of altered ribosomal proteins [22]. Moreover, in many cases, revertants had one or two proteins missing. Several erythromycin dependent mutants were isolated and each generated ribosomal protein (r-protein) lacking mutants. Some kasugamycin dependent mutants exhibited the same property, while the one spectinomycin strain gave rise to mutants lacking large subunit protein L 1 [ 19].
Materials and methods
Identification of mutants
These were generally as described in [6, 12, 13]. Other methods are described in the articles cited. Results Whereas spontaneous streptomycin-dependent mutants may readily be obtained, strains requiring other antibiotics for growth only arise after mutagenesis. ConTable I. Ribosomal proteins missing in mutants of E coil
Protein
Mutant designation
LI LI I Li5 LI9 L24 L27 L28 L29 L30 L33 SI $6 $9 S13 S 17 $20
RDI9, MVI7-10 AM68, AM76, AM77 AMI6-98 AMI49 AM290 AMI25 AM81, AM 108 AM111 AMI0 AM90, AM 108 VTS03 AM80 AM83 AM109-113 AM 111 VT514
Pheno~pe Reference cs ts cs cs cs cs ts ts
[26] [27] [28] [28] [29] [22] [22] [221 [22] [22] [251 unpublished [22] unpublished [22] [25]
Ribosomal proteins were initially resolved on twodimensional polyacrylamide gels [23], stained with amido-black. A protein spot that was apparently missing in the gel of a mutant may simply mean that it had altered mobility and co-migrated with another protein. To determine that such was not the case, a battery of immunological tests were performed. After initial screening with Ouchterlony double diffusion gels, additional tests were made using a modified immuno-electrophoresis technique on cellulose acetate gels. This had the advantage that antibodyantigen complexes did not have to give rise to an insoluble precipitate for cross-reacting material to be detected. Subsequently, immunoblotting has been used; material resolved in SDS slab gels was blotted onto nitrocellulose filters and these were treated with antiserum before immuno-peroxidase staining [24].
Ribosomal proteins missing in mutants of E coli As shown in table I, mutants with any one of sixteen of the fifty-five ribosomal proteins have been found. In every case except that of protein S1 the initial identification was on the basis of two-dimensional polyacrylamide gel electrophoresis (2--D PAGE), with subsequent confirmation by immunological methods. Again with the single exception of protein S 1, protein cross-reacting material (crm) undetectable on the ribosome was also not detected in extracts from whole cells. Therefore, it was not simply a case of the protein being more easily washed off the ribosome. The S1 mutant was found using solely immunological methods, and whereas there was no crm on the ribosome there was crm in whole cell extracts.
Ribosomal protein lacking mutants
Nature of the mutation in r-protein-lacking strains Genetic analysis of r-protein-lacking mutants revealed that in all mutants except S1, the lesion responsible was close to and probably within the gene for the protein which was missing. Apart from protein L24, all proteins found to be lacking have genes either near the end of large transcription units (eg S17, L15, L29, L30) or else, genes that are part of small transcription units (eg $9, $20, L1, L11, L19, L30) [30]. Southern blots from a number of mutants indicated that the DNA fragment bearing the relevant gene was not smaller in the mutant than in a wild-type (E Dabbs and N Fiil, unpublished). Therefore, it was improbable that deletions were responsible for the observed loss, although it could not be excluded that small deletions had occurred which changed the phase of the reading frame. Where there was a size change in the DNA fragment, it was larger than in wild-type. Some of the mutant phenotypes may be due to insertion sequences, as has been found for certain other ribosomal mutants [311. In one case, the nature of the lesion causing an rprotein to be lacking has been determined by. DNA sequencing this being an rplX mutation causing the loss of prot,:in L24 [32]. An AAA codon had changed to a TAA codon at position 61 in the gene, so instead of a lysine codon there was a stop codon [33]. As a result of the base substitution, a 20 amino-acid peptide should be generated instead of the 104 residue wildtype protein L24. A 20 amino-acid peptide should be detectable with the immunological techniques employed. The fact that it was not would suggest that the fragment is rapidly degraded. The rplX lesion conferred a temperature-sensitive conditional lethal phenotype on the cell, which permitted the selection of temperature-resistant revertants. DNA from three revertants was analysed and it was found that the TAA codon had mutated to GAA, TCA, or TTA [33]. Therefore, instead of an ochre stop codon a glutamic acid, serine or leucine residue would be present. Consistent with this, 2-D PAGE of r-proteins from these revertants revealed each possessed a full size protein L24 but of a form electrophoretically distinct from wild-type.
Phenotype arising from r-protein-lacking mutations Generally the lesions responsible for a protein being missing did have an effect on phenotype, as was seen when P1 transduction was used to transfer the mutation out of the strain in which it was isolated. This reduced the likelihood that phenotypes were due to other mutations arising from the mutagenesis employed before selection of the original dependent strain.
641
In all mutants except those lacking L29 or L30, the generation time was significantly increased. Additionally, there was sometimes either a temperature-sensitive (S17, $20, L24) or a cold-sensitive ($9, L15, L27, L28, L33) conditional lethal phenotype. Selection for reversion of the conditional lethal phenotype resulted in strains in which the missing protein was frequently restored: this was investigated for the S 17, L15, L24, L27 and L28 mutants and found to be the case (E Dabbs, unpublished). Such findings were further evidence that the original lesions responsible were not deletions in the genes for these proteins.
Lacking mutants as tools to investigate the ribosome Mutants with individual ribosomal proteins missing offer a simple tool to investigate the role of these proteins. Ribosomes of these strains provide a ready source of intact ribosomes with all proteins except one present at normal stoichiometry. They avoid the need to employ laborious fractionation and reconstitution procedures to arrive at the same end. These mutants have been used as a tool in investigating ribosome topography, function and assembly.
Ribosome topography As stated earlier, all r-protein-lacking mutants have been studied using immt~nological techniques. Many have then been used in experiments to determine the position of individual proteins on the ribosomal surface. Early work on ribosome topography had in some cases led to misidentification of antigenic determinants, which may have reflected non-specific antibody binding or else the presence in purified protein fractions used to raise antibodies of trace amounts of highly immunogenic contaminating material. To avoid misidentification of epitopes on the subunit surface, mutants with individual proteins missing provide an ideal control. This is because any binding sites present in both wild-type and mutant must be due to non-specific binding, whereas sites seen in electron micrographs of subunits from the wild-type but not the mutant very probably are true sites for the protein in question. The location of a protein in particles from wild-type and from a mutant in which the protein missing had been added back was invariably indistinguishable. It was found that the missing protein was rapidly and stoichiometrically incorporated into subunits from the mutant when it was added to a suspension of these particles. This reconstitution occurred under a wide range of conditions, with no special procedure or buffer being necessary. Mutants which have been employed as part of the topographical pinpointing have included one in which proteins L l, L I 1, L I5, LI9 or S17 were missing [34, 35].
642
ER Dabbs
Functional changes in mutant strabts Mutants lacking a particular-protein are an ideal tool to explore the function of that protein. Except for the missing protein, all others are present in the ribosome at the same stoichiometry as in wild-type. The selection which generated strains deficient in a particular protein was spontaneous, which minimises the risk that the altered properties are due to additional lesions not detectable on the basis of changes in 2-D gel analysis. Furthermore, since the missing protein can be readily restored to ribosomal particles, then by titrating back the protein and at the same time monitoring ribosomal properties one can confirm that the altered property is result of the absence of that protein. Mutants lacking protein L I were the first to be studied for changes in organelle function. Ribosomes from such mutants had only about half the in vitro polypeptide synthetic activity of wild-type, but this could be restored to wild-type levels by the addition of purified protein L I to the ribosome suspension prior to assaying activity [36]. In subsequent experiments, it was shown that both the binding of N-acetylPhe-tRNA to ribosomes and the 6-8 fold stimulation of the elongation factor G (EF-G)-dependent GTPase reaction by mRNA together with tRNA were greatly reduced when large subunits from L I mutants were used [37]. A supplement of purified protein L1 again restored these activities to wild-type levels. The basic non-mRNA and non-tRNA dependent EF-G GTPase was not diminished in assays using the mutant ribosomes. This work led to the conclusion that protein L 1 is involved in the interaction between ribosomes and peptidyl tRNA in the peptidyl site [37]. Mutants lacking protein L II have been used to explore the binding to the ribosome of factors in translation. Specifically, ribosomal particles from these strains have been found to have changes in the binding of release factors 1 and 2 [38]; addition of protein L I 1 to mutant ribosomes again resulted in a return to the wild-type pattern of release factor binding. This was consistent with reconstitution experiments that were used to identity ribosomal components involved in factor binding [39]. These L I 1 lacking strains have also been investigated in terms of the subunit interaction. It was found that the mechanism of association of mutant 50S with 30S subunits, or the structure of the resulting 30S-50S couples, is altered in such a way as to cause ejection of an AcPhe-tRNA molecule prebound to the 30S subunits in response to polyU [40]. 30S ribosomal subunits from a strain in which protein $20 was missing were found to be defective in two respects [41]. They have a reduced capacity for association with the 50S ribosomal subunit which results in the impairment of most of the functions requiring a coordinated interaction between the two subunits. Also, they are defective in functions which
do not require their interaction with the large subunit, such as the formation of tertiary complexes with aminoacyl-tRNAs and templates, including the formation of 30S initiation complexes with f Met-tRNA and mRNA. Experiments on other mutants are in progress. In some cases, proteins about which very little is known beyond their sequences, such as L28 and L33, have a much more drastic effect when they are absent than a protein which has been ascribed an important role in the ribosome (LI5). Strains lacking L28 or L33 should help clarify what the part is these proteins play in the ribosome. Subunit assembly in r-protein mutants Most of the proteins for which mutants missing them have been isolated are not ones which are involved in early stages of subunit assembly [4, 5], This is in agreement with the observation that most of these mutants show no abnormalities or defects in assembly. However, two large subunit proteins which have been attributed important roles in organelle assembly, L I5 and L24, are amongst those proteins for which mutants lacking them have been isolated. Protein L24 is, together with L3, one of the two initiator-proteins which first binds to the large subunit [42]. L24 is essential for early large subunit assembly, but is dispensable in later steps and for organelle function [43]. Strains carrying the rplX mutation which leads to absence of protein L24 have a much reduced growth rate, about six-fold slower than wild-type at permissive temperatures. The subunit profile at 30°C reveals a considerably diminished large subunit peak [32] which moreover migrates at 47S rather than 50S [29]. The strain is also temperature-sensitive. In the standard two-step reconstitution, large subunits from L24- mutant showed that - as expected - only one initiator-protein was effective. When the temperature of the first step was reduced from 44 to 26°C however, active particles were reconstituted at about 50% efficiency even though L24 was not present. With this change in conditions, there were again two initiatorproteins. This meant that an additional protein, identified as L20 [44] was present which could replace L24 as initiator of assembly at the lower temperature. This in vitro situation parallels the in vivo situation, in that the L24 lacking mutant can grow at the low end of the normal E coli range of growth temperature. In contrast, strains containing the rplO mutation leading to the absence of protein L15 grow well and ribosomal subunit profiles are similar to that of wildtype, yet protein L 15 has been attributed a pivotal role
Ribosomal protein lacking mutants
in 50S assembly [5]. The homologue of protein L15 in a Bacillus strain does not occupy the same important position in assembly and it may be that E coli possesses alternative pathways which circumvent the requirement for-protein L15 [45].
Changes in gene expression There are at least twenty-five transcriptional units in E coli which give rise to products involved in the translation machinery of the cell [46]. Notwithstanding this plethora, there is coordination in their expression such that balanced synthesis of the components occurs. Mutants lacking individual r-proteins have been used to investigate regulation of this expression. Immunological [27] and pulse labelling [47] studies have been made on mutants lacking protein L1. Both experiments have shown that these strains have elevated levels of protein L11 but not any other ribosomal protein. L11 mutants, on the other hand, have levels of protein L I which are if anything less than normal [27]. The data are fully consistent with in vivo studies which have indicated that the expression of the L l l - L I transcriptional units is regulated by protein LI but not by protein LI 1" when protein L1 is absent from a cell, then increased synthesis rates and increased intracellular levels of protein L 11 would be anticipated.
Ribosomal protein homologies in different organisms Mutant ribosomes can readily incorporate the missing protein when this is added to a suspension of the particles. This incorporation occurs irrespective of whether purified protein or a mixture of ribosomal proteins is added. Moreover, it is found that there is incorporation even if the total ribosomal protein (TP) of an organism other than E coli is used. If there is prior incubation with excess TP from E coli, however, then incorporation of heterologous proteins does not Occur.
These observations have been used as a method of identifying ribosomal correspondences between evolutionarily remote organisms. It has the virtue that no prior fractionation and purification of individual proteins and sequencing or immunology is necessary. This method has been used to identify the B subtilis protein which corresponds to E coil protein L25 [48], and the archaebacterium Methanococcus vannielii protein which corresponds to E coli protein L1 (G St~ftier, personnal communication).
Why do r-protein-lacking mutants arise? As stated earlier, these mutants are generated in several selections. Mutants dependent on different antibiotics in some cases give rise to revertants with lesions in the same r-protein gene. Thus $20- mutants are generated as suppressors of both streptomycin and
643
erythromycin dependence [8, 22]. L l- strains occur amongst the revertants of erythromycin, spectinomycin, or kasugamycin dependents [19, 21, 22]. There are differences, since only erythromycin mutants generate strains missing any of the 16 proteins (table I). However, the parallels between the different selections suggest there must be elements in common. As an approach to understanding what this might be, a comparison was made of two mutants which had the same dependent phenotype but differed in that one generated revertants which included strains with proteins missing from the ribosome and one which did not [49]. Both parental strains were dependent on kasugamycin for growth. The results of experiments in which the two strains were deprived of antibiotics are shown in figure 1. For both, antibiotic removal was bacteriocidal. They both differed therefore from streptomycin dependent strains, where antibiotic removal was bacteriostatic. The two differed from each other in initial response to antibiotic removal: one mutant reacted to this by a surge in growth above that of cells maintained in the presence of kasugamycin. This was paralleled by a surge in protein synthesis [49]. The other mutant showed no such surge. The former strain was one which could be used to select r-protein-lacking mutants, whereas the latter could not. The data showed that the former mutant was dependent on kasugamycin because this antibiotic was needed to depress the level of protein synthesis. Removal led to enhanced protein synthesis, but this enhancement presumably resulted in a lethal disequilibrium within the cell: this was manifested in a cessation of cell growth and in cell death. Therefore, what sort of mutational event could rescue such a strain from the bacteriocidal effect of antibiotic removal? One class of events would be ribosomal mutations which slow dowr, the function of this organelle. This is in agreement with the observation that most lesions leading to an r-protein being missing cause a distinct slowing of growth. Presumably the main way in which ribosome function may be impaired is by loss of one of its consistuent proteins.
Other aspects Two additional uses to which r-protein-lacking mutants can be put are to clone r-protein genes and for crystallographic studies of the ribosome. As stated above, many of the lesions in these mutants generate a conditional lethal phenotype. Therefore, one might be able to clone the corresponding gene based on complementation of this phenotype. The cloning of rplX from E coli wild-type and pseudorevertant strains was done on this basis since the rplX lesion resulting in the absence of protein L24 conferred temperature sensitivity [33].
644
ER Dabbs Mutants with a protein missing from the ribosome can be used to facilitate the incorporation of this protein, to which a metal cluster has been attached (A Yonath, personal communication). Different heavy metal derivatives will be used to permit solution of Xray diffraction patterns, and hence aid in determining the structure of ribosomal particles.
A
Implications o f r-protein-lacking m u t a n t s
0'01t ,
I
,
,
0
2
4
6
TIME(HR)
B
1.0
Over one quarter of the E coli r-proteins have been found to be missing in at least one mutant. There is no reason to suppose that the proteins given in table I represent an exhaustive list of those proteins which are not essential because as long as additional revertants from the appropriate selections were analysed, additions were made to the list. Notwithstanding this, it seems likely that a subset of proteins is essential. One evidence of this may be reflected in the distribution of chloroplast r-protein genes between those that are nuclear encoded and those that are encoded by the chloroplast [50]. There is a strong correlation between the proteins identified as lacking and the chloroplast homologues that are nuclear encoded. Perhaps the set of essential proteins are the ones still encoded by the chloroplast chromosome, as well as of course being absent from table I.
References
o0 . ~ co C3 O
1
0.01
I
0
I,
2
I
t.
I
6
TIME (HR) Fig 1. Growth curves of strains MVIOI and PB67, with viable counts and OD 650 nm. A. MVI01 absorbance in presence ( o ) or absence ( • ) of kasugamycin; cell number in presence ((~) or absence (0) of antibiotic. B. PB67 absorbance in presence (D) or absence ( I ) of kasugamycin; cell number presence (A) or absence (&) of antibiotic. Arrows indicate time at which cells were transferred from antibiotic containing medium (50 l.tg/ml) to fresh medium. Based on data from [48].
1 Wittmann HG (1985) ln: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer (3. eds) SpringerVerlag. NY, 1-27 2 Higo K, Otaka E. Osawa S (1982) Mol Gen Genet 185. 239-246 3 Wittmann-Liebold B (1985) ln: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, NY, 326-361 4 Nomura M, Held WA (1974) ln: Ribosomes (Nomura M, "Tissi~re A, Lengyel P, eds) Cold Spring Harbor Laboratory, 193-223 5 R6hl R, Nierhans KH (1982) Proc Natl Acad Sci USA 79, 729-733 6 Pepersberg W, Geyi D, Hummel H, BOck A (1980) in: Genetics and Evolution of RNA Polymerase, tRNA, and Ribosomes (Osawa S, Ozeki H, Uchida H, Yura T, eds) University of Tokoy Press, 359-377 7 Isono S, Isono K, Hirota Y (1978) Mol Gen Genet 65, 15-20 8 Dabbs ER (1978) Mol Gen Genet 165, 73-78 9 Cundliffe E, Dixon P, Stark M, StUffier G, Ehrlich R, St6ffler-Meilicke M, Cannon M (1979) J Mol Biol 132, 235-252 10 Wienen B, Ehlrich R, St6ffler-Meilicke M, St6ffler G, Smith J, Weiss D, Vince R, Pestka S (1979) J Biol Chem 254, 8031-8041 11 Gorini L (1971) Nature New Bio1234, 261-264 12 Dabbs ER (1977) Mol Gen Genet 158, 55-61 13 Dabbs ER (1976) Mol Gen Genet 149, 303-309 14 Okuyama A, Tanaka N (1975) J Antibiotics 28, 903-905
Ribosomal protein lacking mutants 15 Wallace BJ, Tai PC, Davis BD (1973) J Mol Biol 75, 391--400 16 Wallace BJ, Tai PC, Davis BD (1974) Proc Natl Acad Sci USA 71, 1634-1638 17 Pestka S (1974) AntAgents Chemother 5, 255-267 18 Palmer E, Wilhelm JM (1978) Cell 13, 329-334 19 Dabbs ER (1977) Mol Gen Genet 151,261-267 20 Dabbs ER (1978)JBacteriol 136, 994-1001 21 Dabbs ER (1979)JBacteriol 139, 1072-1074 22 Dabbs ER (1979) J Bacteriol 140, 734-737 23 Kaltschmidt E, Wittmann HG (1970) Analyt Biochem 36, 401--412 24 St6ffler-Meilicke M, Dabbs ER, Albrecht-Ehrlich R, StSffler G (1985) Fur J Biochem 150, 485--490 25 Dabbs ER, Hasenbank R, Kastner B, Rak KM, Wartusch B, St6ffler G (1983) Mol Gen Genet 192, 301-308 26 Dabbs ER, Ehrlich R, Hasenbank R, Schroeter BH, StOffler-Meilicke M, St6ffler G (1981) J Mol Biol 149, 553-578 27 St6ffler G, Dabbs ER, Hasenbank R (1981) Mol Gen Genet 181,164-168 28 Lotti M, Dabbs ER, Hasenbank R, Stt~ffler-Meilicke M, St6ffler G (1983) Mol Gen Genet 192, 295-300 29 Herold M, Nowotny V, Dabbs ER, Nierhaus KH (1986) Mol Gen Genet 203, 281-287 30 Gourse RL, Sharroek RA, Nomura M (1985) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, NY, 766-788 31 Schnier J, lsono S, Cumberlidge G, lsono K (1985) Mol Gen Genet 199, 265-270 32 Dabbs ER (1982) Mol Gen Genet 187,453--458 33 Nishi K, Dabbs ER, Schnier J (1985) J Bacteriol 163, 890--894
645
34 St6ffler G, Cundliffe E, StOffler-Meilicke M, Dabbs ER (1980) J Biol Chem 255, 10517-10522 35 St~iffler G, Noah M, St6ffler-Meilicke M, Dabbs ER (1984) J Biol Chem 259, 4521--4526 36 Subramanian AR, Dabbs ER (1980) Fur J Biochem 112, 425--430 37 Sander G (1983) JBiol Chem 258, 10098-10103 38 'Fate WP, Dogni NJ, Noah M, St/Sffler G (1984) J Biol Chem 259, 7317-7324 39 Tate WP, Schulze H, Nierhaus KH (1983) J Biol Chem 258, 12816-12820 40 G6tz F, Fleisher C, Pon CL, Gualerzi CO (1989) Eur J Biochem 183, 19-24 41 G6tz F, Dabbs ER, Gualerzi C (1990) Biochim Biophys Act 1050, 93-97 42 Nowotny V, Nierhaus KH (1986) Proc Natl Acad Sci USA 79, 7238-7242 43 Spilimann S, Nierhaus KH (1978) J Biol Chem 253, 7047-7050 44 Franceschi FJ, Nierhaus KH (1988) Biochemistry 27, 7056--7059 45 Franceschi FJ, Nierhaus KH (1990) J Bioi Chem 265, 16676--I 6682 46 Nomura M, Gourse R, Baughman G (1984) Annu Rev Biochem 53, 75-117 47 Jinks-Robertson S, Nomura M (1981) J Bacteriol 145, 1445-1447 48 Dabbs ER (1983)JBacterioi 156, 966-969 49 Dabbs ER (1983)JBacteriol 153, 709-715 50 Subramanian AR, Smooker PM, Giese K (1990) In: The Ribosome, Structure, Function, and Evolution (Hill WE, Dahlberg A, Garrett RA, Moore PB, Warner JR, eds) American Society for Microbiology, Washington, 655--663
