Proc. Nati Acad. Sci. USA Vol. 79, pp. 3565-3569, June 1982 Genetics

Nucleotide sequence of the SUF2 frameshift suppressor of Saccharomyces cerevisiae (proline tRNA/branch-shift mutagenesis/nontriplet mRNA decoding/6 sequence) CLAUDIA M. CUMMINS*, THOMAS F. DONAHUEt, AND MICHAEL R. CULBERTSON* *Laboratones of Genetics and Molecular Biology, University ofWisconsin, Madison, Wisconsin 53706; and tSection of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 Communicated by Oliver E. Nelson, February 8, 1982 ABSTRACT To elucidate the molecular mechanism offrame- altered mRNAs produced in strains carrying these suppressible shift suppression by the SUF2 gene ofyeast, the sequences ofDNA contain a 5'-CCCU-3' four-base sequence in place fragments carrying the SUF2-1 and suf2 + alleles of the gene and ofawild-type 5'-CCU-3' proline codon (9). Despite the fact that surrounding regions have been determined. Comparison of the these his4 contain the same codon change at different suppressor and wild-type sequences indicates that the SUF2 gene positions in the his4 message, they are differentially suppressed product is a proline tRNA. Disregarding possible base modifica- by the six suppressors mentioned above. Two of the suppres- tions, we find that the wild-type suf2 + anticodon of the tRNA in- sors, SUF2 and SUF10, suppress both mutations whereas the ferred from the DNA sequence is 3'-GGA-5'. The SUF2-1 mu- other four suppressors, SUF7, SUF8, SUF9, and sufll, fail to tation represents the of a G-C base pair at a position in suppress his4-712 (7). The molecular basis for this division of the gene that corresponds to the anticodon loop of the tRNA. Re- placement ofthe wild-type suf2 + anticodon bya 3'-GGGA-5' four- the suppressors is unknown. base anticodon enables the SUF2-1 tRNA to suppress the 5'- A general molecular cloning protocol based on complemen- CCCU-3' four-base codons generated as the result ofthe his4-712 tation in yeastfollowing transformation (10) has been specifically and hi&4-713 frameshift mutations. This nontriplet codon-anticodon adapted to clone DNA fragments carrying the yeast frameshift interaction restores the correct readingframe andallows synthesis suppressor and has been used to isolate a 10.7-kilobase of a functional his4 . fragment carrying the SUF2 suppressor gene (8). Three kinds of evidence support the conclusion that the cloned DNA frag- Frameshift ihutations result from addition or of base ment carries the SUF2 gene. Plasmids carrying the fragment pairs in a gene encoding a protein product. Mutations of this confer suppression of SUF2-suppressible frameshift mutations type usually render the gene product nonfunctional due to in- when introduced into yeast by transformation. Appropriate ability of the translational apparatus to recognize the shift in plasmids carrying the fragment integrate by homologous re- readingframe. This results in an incorrect specification ofamino combination at the SUF2 locus. In addition, the fragment shares acids and in termination of at the first out-of-phase a homologous segment of DNA with yeast DNA contained in nonsense codon encountered. the plasmid pYe98F4T. This plasmid is known to carry the The correct can be restored in strains carrying CDC10 gene, which maps 1 centimorgan from the SUF2 locus frameshift mutations by various compensatory mechanisms. In near the chromosome III centromere (8, 11). some instances, suppressor mutations in genes external to that The genetic properties ofSUF2 and the implication ofaltered which carries the frameshift have been shown to affect tRNAs in frameshift suppression led us to screen for the pres- the structures of tRNAs, tRNA base-modification enzymes, or ence of tRNA genes on the clone. Hybridization of restriction ribosomal (1-3). Altered tRNAs containing an extra fragments from the SUF2 clone to 4S RNA revealed the pres- base in the anticodon have been implicated in suppression of ence ofat least two tRNA genes located on noncontiguous frag- + 1 frameshift mutations (1). Although such tRNAs may be ca- ments within the 10.7-kilobase segment. These two fragments pable ofreading four bases rather than the normal three bases, were subcloned. One ofthe two resulting plasmids was capable the exact mechanism governing four-base translocation on the of transforming an appropriate yeast recipient to a suppressor ribosome is uncertain (4). In the case of altered tRNA modifi- (8). It remained to be determined whether the tRNA cation enzymes and ribosomal proteins, the molecular mecha- gene carried on this restriction fragment was synonymous with nisms of frameshift suppression are unknown. the SUF2 gene. A complete molecular analysis of nontriplet decoding inter- In this communication we report the results of DNA se- actions, typified by suppression offrameshift mutations, can be quence analyses of restriction fragments carrying the SUF2-1 expected to provide a more detailed view of the normal in vivo suppressor mutation and the wild-type suf2 + allele ofthe gene. decoding mechanism and of the mechanisms for translational The DNA sequences show that the SUF2-1 suppressor phe- control of protein synthesis. To examine this problem in the notype results from insertion of a G-C base pair within a gene lower eukaryote Saccharomyces cerevisiae, mutationally in- encoding a proline tRNA. The implications for tRNA-mRNA duced nontriplet reading systems have been developed in our interactions resulting in suppression of frameshift mutations laboratory by the isolation ofexternal suppressors offrameshift are discussed. mutations at the his4 locus. Suppressor mutations mapping at 25 different loci have been identified (5-8). MATERIALS AND METHODS Six of these suppressors have been shown to suppress the Enzymes and Chemicals. [a-32P]dNTPs (840 Ci/mmol; 1 Ci +1 G-C insertion mutations his4-712 and his4-713 (7, 9). The = 3.7 x 10'° becquerels) were purchased from New England Nuclear. Restriction enzymes and T4 DNA ligase were products The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations:bp, basepair(s);ICR-170, 2-methoxy-6-chloro-9[3-(ethyl- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2-chloroethylamino)propylamino]acridine. 3565 Downloaded by guest on September 29, 2021 3566 Genetics: Cummins et aL Proc. Natl. Acad. Sci. USA 79 (1982) of Bethesda Research Laboratories. DNA polymerase I was The presence of these restriction sites was confirmed by sub- purchased from Sigma, and DNase I was from Worthington sequent DNA sequence analysis. In addition, a third Xba I site Biochemicals. DEAE-cellulose (DE-52) was purchased from was revealed in close proximity to the terminal Pst I site. The Whatman. Avian myeloblastosis virus RNA-dependent DNA strategy for analysis of the DNA sequence from the most left- nucleotidyltransferase (reverse transcriptase) was obtainedfrom ward Xba I site to the rightward Xho I site is shown in Fig. 1. the Division ofCancer Cause and Prevention, National Cancer The sequence was determined for both strands of the 867-bp Institute. Xba I/Xho I fragment. Plasmids. Plasmids pCC5 and pCC6 are subclones of pre- The SUF2 Gene Encodes a Proline tRNA. The DNA se- viously described plasmids, pCC1 and pCC4 (8). To construct quence of the suf2' allele on pCC6 is shown in Fig. 2. When pCC5, the Pst I/HindIII fragment ofYIp33 (12) was removed this sequence was compared with the corresponding sequence and replaced with a Pst I/HindIII fragment from pCC1 that ofthe Xba I/Xho I fragment derived from pCC5, which carries carries the SUF2-1 allele. Similarly, pCC6 was generated by the SUF2-1 allele, the two sequences were found to differ at a replacing the Pst I/HindIII fragment of YIp33 with a Pst I/ single position. An autoradiogram of a portion of a gel encom- HindIII fragment from pCC4 that carries the suf2' allele. passing the site ofthe mutational difference is shown in Fig. 3. DNA Preparation. Plasmid DNA was prepared according to The evidence provided by this gel supports the conclusion that the method of Hicks and Fink (13). [a-3zP]dATP was incorpo- the SUF2-1 suppressor phenotype results from insertion of a rated into plasmid DNA by nick-translation with DNA poly- single G-C base pair in the DNA. This resultwas not unexpected merase I (14). DNA was transferred from agarose gels to nitro- considering that the SUF2-1 allele was induced by using the cellulose filters by using the method of Southern (15). mutagen 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethylamino)- DNA Sequence Analysis. DNA fragments to be analyzed propylamino]acridine dihydrochloride (ICR-170), which is were labeled at their 3' ends by using reverse transcriptase and known to cause G-C base-pair insertions in the DNA of both [a-32P]dNTPs as described by Smith and Calvo (16). The se- prokaryotes (20) and eukaryotes (9). quence of the DNA was determined by the method of Maxam By examining the DNA sequence in the regions surrounding and Gilbert (17). In some cases, 15 ,ul ofglacial acetic acid was the site ofthe + 1 G-C base-pair insertion, we found that a RNA substituted for 5 M NaCl in the cytosine-specific reactions (18). sequence complementary to one ofthe two DNA strands could be drawn that has features characteristic of tRNAs. In partic- ular, the RNA sequence deduced from the DNA sequence can RESULTS be folded into the clover-leaf secondary structure typical ofall Subeloning, Restriction Mapping, and Strategy for Analysis tRNAs whose sequences have thus far been determined (Fig. of the DNA Sequence. A restriction map of the 10.7-kilobase 4). In addition, various constant regions found among different BamHI fragment originally found to carry the SUF2-1 allele of tRNAs are present at their appropriate positions in this RNA the SUF2 gene is shown in Fig. 1. Subsequent analysis dem- sequence. On the basis ofthe secondary structure proposed in onstrated that the Pst I/Xho I fragment shown as a hatched re- Fig. 4 for the tRNA, the site of the mutational difference be- gion in the figure contains a functional SUF2 gene (8). In prep- tween the SUF2-1 and suf2+ DNA sequences is located in the aration for DNA sequence analysis, the larger Pst I/HindIlI anticodon ofthe corresponding tRNA gene product. The wild- fragment, which includes the SUF2-1 allele, was subcloned into type 3'-GGA-5' anticodon triplet in the suf2+ tRNA is replaced the plasmid YIp33 (12), resulting in the plasmid designated by the 3'-GGGA-5' quadruplet in the SUF2-1 tRNA. pCC5. For purposes ofcomparison, a DNA fragment carrying The sequence analyses indicate that the wild-type DNA en- the wild-type suf2+ allele was cloned by using aprotocol similar codes 72 ofthe proposed 75 bases in the gene product. The ter- to that described by Roeder and Fink (8, 19). The corresponding minal C-C-A sequence found at the 3' end ofall tRNAs is not Pst I/HindIII fragment carrying the suf2+ allele was subcloned encoded in the gene and is therefore presumed to be added to into YIp33, resulting in the plasmid designated pCC6. All fur- the gene product post-transcriptionally by a tRNA nucleoti- ther analyses were carried out with appropriate restriction frag- dyltransferase. The presence ofan intervening sequence in the ments derived from pCC5 (SUF2-1) and pCC6 (suf2+). gene can be excluded by examination ofthe secondary structure The Pst I/Xho I fragment carrying the SUF2 gene was found of the tRNA inferred from the DNA sequence. The DNA se- to contain a single restriction site for the enzyme Ava I. Three quences oftRNA genes containing intervening sequences have bands of e230, 270, and 360 base pairs (bp) could be resolved additional nucleotides in the region corresponding to the an- after Xba I digestion, indicating the presence oftwo Xba I sites. ticodon loop (21). BXRPst Pst R P p Pst x HX H H B I H PPstB I aII II i -.LAAAdr H I

XboI Avol Xbol XbaI Xho I

I II I I I_I ______I_ I IL2I ;II aI I I . I I I I I I I I I I I 1 0 10U1 ^^ 2000%^O% 3V20 400A ^^ 500&^^ &%6VU 7(V VUotN^ 1_ bp

FIG. 1. Strategy used and relationship of the fragment analyzed to the originalBamHI fragment found to carry the SUF2 frameshift suppressor (8). Restriction sites: B, BamHI; X, Xho I; R, EcoRI; P, Pvu II; Pst, Pst I; H, Hindl; Bgl, Bgi II. !2, Fragments known to carry tRNA genes (ref. 8; unpublished data). The position of the SUF2 tRNA gene is shown in black with an arrow indicating the direction of of the gene; other arrows indicate the direction and extent of analysis from each restriction site. Downloaded by guest on September 29, 2021 Genetics: Cummins et al Proc. Natd Acad. Sci. USA 79 (1982) 3567

TCTAGAAATCATAATCTGTACCTCCATTCAGCCATCTGAGGAACCTCcGAAAT MCGAGTMATGTTCAAACAT GCCTTGGTTTATTGTACGA 100 AGsATC-MAGTA'TTAGACATGGAGGTAAGTCGGTAGACTCcTTGGAGGCTTTATTGCTCATTTACAGMmGTACGGTACGCGGAACCAAATAACATGCT XbaI Odel GTMTAGCATATTGC.AAIAACTGCTCCTGAGTACACTTGTMACGTCGGMAGGAAAATGCCGTA'CCATTTCTGCCAGTAGCGACACCACACATT 200 CMAATCGTATAACGITT TIIIIGACGAGGACTCATGTGAACAAAATGCAGCCMCTAMACGGCATGGTAAAGACGGTCATCGCTGTGGTGTGTM Ddel

GTAAAAACAAT ThCCMTTGAAAAATCC 300 CA A I UIU ITTTAGG HI q AvaTAval I DdF AITnW X6I9F

CGGCAGAACAGCGCCTGAAGTCTGGGATACAGCCCTATMTCCCTGCCATCGTCATTGACCIZ|W CTTAACGACCAGA'TAACAGCCAGTTATTGMAAG 400 GCCGTCTTGTCGCGGACTTCAGACC'CTATGTCGGGATAAAAGGGACGGTAGCAGTAA CTGGkAGATTGCTGGTCTAATTGTCGGTCAATAACTTC

MGCGAACGTGAAGTMAAACTGCAAAATG~GTCGTMTAAGTCAAAAGTiAACCCTGCGTCACACATGAGiAACATTATCGCTAAGTTG-rTGTTACTAcTi 500 AAACGCTTGCACTTCATTTTGACGTTTTACCAGCAAAATTCAGTTTTCATTGGGACGCAGTGTGTACTCMGTAATAGCGATTCAACAACAATGATGAADdI-

CTTGTAATTAACTTACTGTCGCATTCCAAATGGACTGCGAAACAGACACGAAACATTACGAAGTGACGACAGAGTTA TATTGTCATTAAAGAGAATG 600 GAACATTAATTGAATGACAGCGTAAGGMACCTGACGCMGTCTGTGCMGTTAATGCMCACTGCTGTCTCAMATAACAGTAMmCTCTTAC

ATGACCTCCG~GGTMAACTGT'GAGMATMGTCTAGACCATTCGTTAAACTTCATCMMTTGAGAAGCATAGTMMAMATMTTMCMGTTMCACMAC 700 TACTGGAGGCCCATTGACACTCTTAT.TCAGATCTGGTMAGCMMGAAGTAGAMTTAACTCTTCGTATCATTTTTTATTMTTGTTCMATTGTGTTG 75FF TAT.TT.C.CA.C.AC.A.GA.AaAG TATCTTGACCCATGCTACCAAGGACATAAGTMTATCCTC'CACCACTTTTACTCAMGTATATCTCATTC2TGAGAAATGGTGAATCTTGAGATAATWT 800 ATAGMACTGGGTACGATGGTTCCTGTATTCATTATAGGAGGTGGTGMMATGAGMCATATAGAGTAGAcT=AcccAoTACAACrcTATTAAcA Dde

TG GATTCCATTGTTGATAAAGGCTATAATATTAGGTATACAGAATATACTAGAAGTTCTCGAG ACCCTAAGGTAACAACTATTTCCGATATTATAATCCATATGTCTTA TATGATCTTCAAGAGGAGCTC HF MnI ThIF FIG. 2. DNA sequence of the suf2+ proline tRNA gene (large letters) and surrounding regions. Transcription of the tRNA gene is from right to left. The fiagment analyzed includes approximately one-third of a 8 element (shown in italics). Base Composition, Open Reading Frames, and Potential by computer analysis. A determination was made ofopen read- Secondary Structure in the DNA Sequence. The tRNA gene ing frames on each strand defined by the positions ofAUG start shown to be synonymous with the SUF2 frameshift suppressor codons and UAA, UAG, and UGA stop codons in all three pos- gene represents a (G + C)-rich region located in an (A + T)-rich sible reading frames. In addition, the DNA sequence was fragment. The (G + C) content ofthe tRNA gene is 67%as com- screened for possible secondary structures. pared with 40% for the entire fragment. This latter value is in The results indicate that the largest possible translation prod- close agreement with the published value of38% for total yeast ucts encoded in the sequence correspond to two open reading DNA (22). Other features ofthe DNA sequence were revealed frames, one starting at position 465 and ending at position 656 and the other starting at position 713 and ending at position 516 a b (Fig. 2). These regions are of sufficient length to encode pro- C G G#A T&C C teins of 64 and 66 amino acids, respectively. Whether these G G#A TIC regions actually encode proteins is not known. The computer search uncovered apotentially interesting sec- ondary structure (Fig. 5). This secondary structure is located G _ _ c at the 3' end ofthe mature tRNA coding region. The stem and loop structure requires base pairing of six nucleotides within C C m_ -Cs_ the structural portion ofthe tRNA gene. The significance ofthis _- _C secondary structure is unknown. However, the stretch of five C m_ mm__ G thymines in the secondary structure is likely to serve as the site C _ A for termination oftranscription ofthe tRNA gene. Such thymine tracks have been found at the 3' ends ofa number ofother genes transcribed by yeast RNA polymerase III and are considered to be transcription termination signals (23, 24). A A Identification of a 8 Element in the Fragment Analyzed. When the Pst I/Xho I fragment was used as a hybridization FIG. 3. Autoradiogram of a DNA analysis gel of the transcribed probe againstrestricted genomic DNA, alarge numberofbands strands of aportionof theSU2- (a)andsuf (b)fallelesoo Comparison were observed in the autoradiograms (figure not shown). It of the sequences shows that the SUF2-1 mutation resulted from in- seemed unlikely that the tRNA gene itself could be reiterated sertion of a GC base pair in the DNA at a position corresponding to in the anticodon loop of a proline tRNA. The nucleotides found in the the genome to such an extent. Therefore, we considered the and wild-typo proline tRNA anticodons are inferred from the possibility that the fragment carrying the SUF2 gene might also DNA sequences of the noncoding strands and are shown in boxes. Note carry an additional sequence that is reiterated in the genome. also the + 1 shift in the sequence ladder beyond the site of mutation. The DNA sequence was screened for the possible presence of Downloaded by guest on September 29, 2021 3568 Genetics: Cummins et aL Proc. Nati Acad. Sci. USA 79 (1982)

a Ao" DNA sequence is located in a 8 sequence (see Fig. 2). The ho- C C mology extends 97 bp from the Xho I site (positions 771-867). C The 8 sequence of the SUF2 DNA fragment differs at eight pG-C G-C positions from those at the ends of TYl-B10 and TYl-D15. This G-C extent ofsequence divergence (8.2%) is consistent with the re- C-G G-C sults of Gafner and Phillipsen (27), who observed that solo 8 U U sequences show a sequence divergence of up to 15%. U GG CCCUGA I I I I I G AG AU C U GG DISCUSSION I ccGGGUU C GGU AU6AUG6 GAU Cu The results presented here define the molecular structure of UC-GGAGG the SUF2 suppressor gene and demonstrate that the gene prod- C-G uct is an altered proline tRNA. In strains carrying the SUF2-1 G-C C-G mutation, the proline tRNA gene contains a + 1 G C insertion U U at a position that corresponds to the anticodon loop ofthe tRNA, UAGGG generating a 3'-GGGA-5' four-base anticodon in place of the b 3'-GGA-5.' anticodon found in wild type. There is no published \ / tRNA sequence in which an adenine residue occupies the wob- ble C-G position in an anticodon; inevitably the adenine residue is U * G deaminated to inosine (28). Although a minor base analysis has C-G not yet been undertaken on the SUF2 gene product, it appears G-C C-G likely that the wild-type anticodon actually contains inosine U U rather than adenine in the third position. If this is the case, it U 6 AG is uncertain whether or not the insertion of an additional gua- GG nine in the SUF2-1 anticodon would interfere with deamination of the adenine in the fourth position. FIG. 4. Secondary structures of the suf2' (a) andSUF2-1 (b) forms of the proline tRNA inferred from the DNA sequences of the wild-type Mechanisms of Frameshift Suppression. On the basis ofthe and mutant alleles. Only the anticodon stem and loop are shown for altered 3'-GGGA-5' (or3'-GGGI-5') anticodon sequence in the the SUF2-1 tRNA; the remainder of the mutant tRNA is identical to tRNA, we propose that the 5'-CCCU-3' four-base codons lo- that shown for the wild type. The 3'-C-C-A nucleotides found at the cated at the his4-712 and his4-713 mutational sites interact with termini of all tRNAs are not encoded in the gene. The base modifi- the anticodon by standard base pairingat all four positions, lead- cations of the wild-type and mutant forms of the tRNA have not yet ing to four-base reading by the tRNA and to suppression ofthe been determined. mutations (Fig. 6). However, from these limited data it would be premature to conclude that standard base pairing at all four two well-characterized sequences, TYl and 8, that have been positions is required.for suppression. Data from our laboratory shown to be reiterated in the genome (25). sequences are indicate that an altered glycine tRNA (SUF16) containing a 3'- =300 bp long and are present in at least 100 copies in the yeast CCCG-5' four-base anticodon can suppress the 5'-GGGU-3' genome. These sequences are sometimes present as solo copies sequence located at the his4-38 mutational site (refs. 5, 9; un- and, in addition, are characteristically found as direct repeats published). The G-U wobble pair at the fourth position does not at the ends ofTYl elements (25, 26). prevent suppression. Thus, standard base pairing or wobble at Comparison ofthe sequence ofthe SUF2 DNA fragment with published 8 sequences found at the ends ofTYl-B1O and TYl- GIn Pro Gly D15 (27) revealed that the Xho I site at one terminus ofthe SUF2 H1S4 5' CAA CCU GGU 3' T T T T his4-712 CAA CCCU GGU T-A SUF2 anticodon 3' GGGA 5' -.suppression to His G-C Ile Thr Pro Glu T-A H1S4 5' AUU ACC CCU GAA 3' T-A A-T his4-713 AUU ACC 'CCCU'GAA C-G SUF2 onticodon 5! GGGA 5' suppression to His A-T Ser Ser Leu Ser C-G H1S4# 5' UCG AGC CUG AGU 3' C T C-G his4-506 UCG AGC TZIJ AGU C-G SUF2. onticodon 3' GGGA 5- nosuppression1t His-or HoI G T FIG. 6. Suppression of ICR-170-induced mutations by SUF2-1. 227 C-G 182 his4-712 and his4-713 are G-C insertion mutations in proline codons T CC C G-G CT TC GC TA CT in the his4 gene (9). The four-base mRNA codons that must be rec- ognized by the SUF2-1 tRNA anticodon to allow suppression to a His' FIG. 5. Potential secondary structure in the DNA sequence flank- phenotype are diagrammed. hi84-506 is a polar his4A mutation. ing the proline tRNA gene. For convenience, the folding of only the SUF2-1 is unable to suppress his4-506 to a His' phenotype. The pres- noncoding strand is shown. The numbers correspond to base-pair num- ence of SUF2-1 is also insufficient to relieve pQlarity into the down- bers assigned in Fig. 2. The six 3'-terminal nucleotides of the proline stream his4C function, as indicated by the inability ofhi&4-506SUF2- tRNA gene (boldface letters) are included in the structure. 1 to grow in the presence of histidinol (Hol-). Downloaded by guest on September 29, 2021 Genetics: Cummins et al. Proc. Nati Acad. Sci. USA 79 (1982) 3569

the fourth position are both compatible with suppression. the different classes of suppressors and suppressible mutations To determine the effect of nonstandard combinations of will be required. Of particular interest will be those suppres- bases on suppression, his4-506 has been tested for suppressi- sors, either in Salmonella or in yeast, that are capable of sup- bility in strains carrying SUF2-1 (unpublished data; see Fig. 6). pressing non-wobble-related frameshift mutations. This his4 mutation results from insertion of a cytosine residue in a his4A 5'-CUG-3' leucine codon, generating the four-base We thank Irving Edelman ofthis laboratory for contributing hts4-506 codon 5'-CCUG-3' (9). Although an altered proline tRNA could SUF2-1 suppression data, Richard Gaber of this laboratory for unpub- potentially recognize the 5'-CCUG-3' sequence, SUF2-1 fails lished data on the SUF16 gene, and Dr. Gerald Fink for generous as- to suppress the mutation to a His' phenotype. The lack of sistance in training C.M.C. in techniques for analysis of DNA se- suppression is not the result of impaired protein function due quences. This research was supported by the College of Agricultural to a substitution of proline for leucine in the amino acid se- and Life Sciences, University ofWisconsin, Madison, and U.S. Public quence; the suppressor is also unable to relieve polarity ofhis4- Health Service Research Grant CA26217 (to M.R.C.). C.M.C. is a 506 into the downstream his4C gene, as indicated by lack of Trainee supported by U.S. Public Health Service Training Grant growth on medium containing histidinol (Hol-) and by the ab- GM07133 in the Department of Genetics. This is Laboratory of Ge- sence of his4C-encoded histidinol dehydrogenase activity. The netics paper no. 2512. GA pair at the fourth position may prevent an interaction lead- 1. Riddle, D. L. & Carbon, J. (1973) Nature (London) New Biol ing to suppression. In addition, a G'G pair at the fourth position 242, 230-234. may also prevent suppression, as indicated by the failure of 2. Atkins, J. (1980) in Transfer RNA: Biological Aspects, eds. Soll, SUF16 (anticodon 3'-CCCG-5') to suppress his4-519 (5'-GGGG- D., Abelson, J. & Schimmel, P. (Cold Spring Harbor Laborato- 3') (unpublished). ries, Cold Spring Harbor, NY), pp. 439-452. In contrast to these results, the sufG and sufj suppressors 3. Gorini, L. (1974) in Ribosomes, eds. Nomura, M., Tissieres, A. in Salmonella (4) and the SUF1, SUF3, SUF4, SUF5, SUF6, and & Lengyl, A. (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY), pp. 791-803. SUF17 suppressors in yeast (5, 9) are able to suppress non-wob- 4. Roth, J. (1981) Cell 24, 601-602. ble-related mutations. For example, these yeast suppressors 5. Culbertson, M. R., Charnas, L., Johnson, M. T. & Fink, G. R. suppress his4-38 (5'-GGGU-3') and his4-519 (5'-GGGG-3'). (1977) Genetics 86, 745-764. According to standard rules for base pairing and wobble, no 6. Culbertson, M. R., Underbrink, K. M. & Fink, G. R. (1980) Ge- anticodon should be able to suppress both mutations. However, netics 95, 833-853. in both Salmonella and in yeast, indirect evidence implicates 7. Cummins, C. M., Gaber, R. F., Culbertson, M. R., Mann, R. & Fink, G. R. (1980) Genetics 95, 855-875. the involvement of tRNA in these examples of suppression. 8. Cummins, C. M. & Culbertson, M. R. (1981) Gene 14, 263-278. Two alternative models have been proposed to explain co- 9. Donahue, T. F., Farabaugh, P. J. & Fink, G. R. (1981) Science don-anticodon interactions in frameshift suppression (see ref. 212, 455-457. 2). One model assumes that the altered tRNAs "read" four bases 10. Beggs, J. D. (1978) Nature (London) 275, 104-109. by standard base pairing or wobble interactions. According to 11. Clarke, L. & Carbon, J. (1980) Nature (London) 287, 504-509. the second model, a tRNA containing an extra nucleotide in its 12. Botstein, D., Falco, S., Stewart, S. E., Brennan, M., Scherer, S., Stinchcomb, D. T., Struhl, K. & Davis, R. W. (1979) Gene 8, anticodon occupies an unusually large space on the mRNA. The 17-24. ribosome responds by translocating four nucleotides along the 13. Hicks, J. B. & Fink, G. R. (1977) Nature (London) 269, 265-267. message without regard for the nature ofthe base occupying the 14. Maniatis, T., Jeffrey, A. & Kleid, D. G. (1975) Proc. Natl Acad. fourth position in the anticodon. Neither model in its simplest Sci. USA 72, 1184-1188. form is completely satisfactory; recent data indicate that some 15. Southern, E. M. (1975) J. Mol Biol 98, 503-517. suppressors appear to violate standard base pairing or wobble 16. Smith, D. R. & Calvo, J. M. (1980) Nucleic Acids Res. 8, 2255-2274. rules and some fourth position mRNA'tRNA base combinations 17. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol 65, have been shown not to be compatible with suppression. 599-560. A third model may be proposed that incorporates features 18. Rubin, C. M. & Schmid, C. W. (1980) Nucleic Acids Res. 8, of both ofthe above models and assumes that certain nonstand- 4613-4619. ard fourth-position base combinations are tolerated whereas 19. Roeder, G. S. & Fink, G. R. (1980) Cell 21, 239-249. others are not. The ease with which different fourth-position 20. Yourno, J. & Heath, S. (1969) J. Bacteriol 100, 460-468. bases in 21. Goodman, H. M., Olson, M. V. & Hall, B. D. (1977) Proc. Natl the tRNA anticodon can fit into the A site of the Acad. Sci. USA 74, 5453-5457. mRNA-ribosome complex may influence which of the fourth- 22. Feldmann, H. (1976) Nucleic Acids Res. 3, 2379-2386. position base combinations can lead to suppression. For ex- 23. Olah, J. & Feldmann, H. (1980) Nucleic Acids Res. 8, 1975-1986. ample, size constraints may preclude purine-purine combina- 24. Koski, R. A., Clarkson, S. G., Kurjan, J., Hall, B. D. & Smith, tions. Less bulkypyrimidine-pyrimidine or possibly nonstand- M. (1980) Cell 22, 415-425. ard purine-pyrimidine combinations may provide a better fit 25. Cameron, J. R., Loh, E. Y. & Davis, R. W. (1979) Cell 16, such that an interaction leading to suppression is permitted. 739-751. 26. Farabaugh, P. J. & Fink, G. R. (1980) Nature (London) 286, To test this model and to determine whether post-transcrip- 352-356. tional tRNA base modifications or message context have an in- 27. Gafner, J. & Phillipsen, P. (1980) Nature (London) 286, 414-418. fluence on the efficiency ofsuppression, further information on 28. Gauss, D. H. & Sprinzl, M. (1981) Nucleic Acids Res. 9, rl-r23. Downloaded by guest on September 29, 2021