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Tetranucleotide Loops GAAA and UUCG in Fission Yeast

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Tetranucleotide Loops GAAA and UUCG in Fission Yeast Proc. Natl. Acad. Sci. USA Vol. 90, pp. 5409-5413, June 1993 Biochemistry Functional interchangeability of the structurally similar tetranucleotide loops GAAA and UUCG in fission yeast signal recognition particle RNA (RNA structure/RNA-protein interactions) DAVID SELINGER, XIUBEI LIAOt, AND Jo ANN WISEt Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801 Communicated by Joan A. Steitz, February 10, 1993 (receivedfor review January 5, 1993) ABSTRACT Signal recognition particle (SRP) RNA exhib- in catalytic and informational RNAs as well (7, 8). This its significant primary sequence conservation only in domain prevalence was previously proposed to arise from their IV, a bulged hairpin capped by a GNRA (N, any nucleotide; R, ability to increase hairpin stability (9) but may instead be a purine) tetranucleotide loop except in plant homologs. Tetra- consequence of their well-defined three-dimensional confor- loops conforming to this sequence or to the consensus UNCG mations (10). Recently, solution structures of small synthetic enhance the stability of synthetic RNA hairpins and have RNAs containing each of these tetraloops have been solved strikingly similar three-dimensional structures. To determine by two-dimensional NMR spectroscopy (11, 12). Despite the biological relevance ofthis similarity, as well as to assess the their different sequences, they adopt quite similar structures relative contributions of sequence and structure to the function in which the first and fourth bases are hydrogen bonded, the of the domain IV tetraloop, we replaced the GAAA sequence in second base has little interaction with the remainder of the fission yeast SRP RNA with UUCG. Haploid strains harboring loop, and the phosphate backbone between the second and this substitution are viable, providing experimental evidence third nucleotides is extended as a consequence of S-type for the functional equivalence of the two tetraloops. We next sugar puckering. tested the two sequences found in plant SRP RNAs at this We previously reported the in vivo effects of point muta- location for function in the context of the Schizosaccharomyces tions in the domain IV tetraloop of fission yeast SRP RNA pombe RNA. While substitution of CUUC does not allow (nucleotides 160-163; wild-type sequence GAAA) (13). Both growth, a viable strain results from replacing GAAA with lethal alleles identified were transversions at G-160, which UUUC. Although the viable tetraloop substitution mutants had been implicated in SRP19 protein binding by RNase exhibit wild-type growth under normal conditions, all three protection studies (14, 15). However, a G at this position is express conditional defects. To determine whether this might also critical to the integrity of the tetraloop, and there is a be a consequence of structural perturbations, we performed strong correlation between the phenotypes of the remaining enzymatic probing. The results indicate that RNAs containing point mutants we examined and predicted perturbations of tetraloop substitutions exhibit subtle differences from the wild the structure. To gain further insight into the role of domain type not only in the tetraloop itself, but also in the 3-base pair IV, as well as to assess the relevance of recent in vitro adjoining stem. To directly assess the importance of the latter structural data to the situation in vivo, we analyzed both the structure, we disrupted it partially or completely and made the effects of en bloc tetraloop substitutions and the conse- compensatory mutations to restore the helix. Surprisingly, quences ofdisrupting and restoring the adjoining stem. Taken mutant RNAs with as little as one Watson-Crick base pair can together, the results of our studies imply that the in vivo support growth. function of this region is determined by its structure. Signal recognition particle (SRP) is an RNA-protein complex EXPERIMENTAL PROCEDURES that targets ribosomes translating presecretory proteins to Materials. Enzymes were purchased from BRL and New the endoplasmic reticulum membrane (reviewed in ref. 1). England Biolabs; mutagenesis reagents were from Amer- The extensively studied canine SRP is composed of six sham; DNA sequencing reagents were from United States polypeptides and one 300-nucleotide RNA (2, 3). SRP RNA Biochemical; RNases were from Pharmacia; and calf intes- (also referred to as 7SL) has been identified in a variety of tinal alkaline phosphatase was from Boehringer Mannheim. organisms (reviewed in ref. 4) and can be folded into a Sequencing primers and mutagenic oligonucleotides were phylogenetically conserved secondary structure consisting of synthesized at the Biotechnology Center at the University of four domains: a short base-paired region at the 5' end (domain Illinois. Radiolabeled [y_32P]ATP was from ICN. I); a long central helix that includes the 3' end (domain II); Site-Directed Mutagenesis. Targeted mutations were intro- and two internal stem-loop structures, one extensively base duced into the cloned SRP7 gene carried on the phagemid paired (domain III) and one containing several internal loops pWEC4.2 (16) by standard methods (17, 18) with the follow- (domain IV) (nomenclature according to ref. 5). The se- ing oligonucleotides: STL1 (5'-ATGTGCATTSC- quence, as well as the secondary structure, of domain IV is GAASAACCTCCATC-3'), replaces nucleotides 159-164 conserved in SRP RNA homologs from bacteria to humans with SUUCGS sequences where S is G or C; STL2 (5'- (4). This helix terminates in a tetranucleotide loop that ATTCCGAAGAACCTCCATC-3'), generates a variant not conforms to the consensus GNRA (N, any nucleotide; R, obtained with STL1; PTL1 (5'-TGTGCATTGGAAR- purine) except in plant SRP RNAs, which have four pyrim- CAACCTCCA-3'), replaces nucleotides 160-163 with the idines at this location. GNRA and UNCG tetraloops are corresponding segment of plant SRP RNA; PTL2 (5'- highly overrepresented in RNAs (6) and are found frequently Abbreviation: SRP, signal recognition particle. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Molecular Biology, Research In- payment. This article must therefore be hereby marked "advertisement" stitute of Scripps Clinic, La Jolla, CA 92037. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 1To whom reprint requests should be addressed. 5409 Downloaded by guest on September 26, 2021 5410 Biochemistry: Selinger et al. Proc. Natl. Acad. Sci. USA 90 (1993) TGTGCATTTGAARCAACCTCCA-3'), places a G-A pair Table 1. Phenotypes conferred by mutations targeted to the adjacent to the plant tetraloops; M3a (5'-ATGTGCAT- domain IV terminal region TG*TT*TCC*AACCTCCAT-3'), creates mutations at posi- Line Allele Sequence Growth OTS tions 159, 162, and 164 (45% degeneracy was allowed at the positions marked with an asterisk); BP2 and -3 (5'- 1 Wild type GGAAAC GATGTGCAT1T2GTTTCCA3A4CCTCCATCG-3'), creates 2 A162C GGACAC Viable + mismatches in the second and third base pairs flanking the 3 G159U/A162C UGACAC Dead tetraloop and changes the A-U pairs to C-G pairs (T1 = 50% 4 G159U/A162U UGAUAC Dead T/50% G, T2 = 50% T/50% C, A3 = 17% A/83% G, and A4 5 A162C/C164G GGACAG Viable +++ = 17% A/83% C); BP-C (5'-TGATGTGCAGCGTTTC- 6 STL1-3 GUUCGC Wild type +++ CGCCC-3'), replaces both A-U pairs with G-C pairs; D4-E 7 STL2-1 CUUCGG Viable ++ (5'-GATGTGCATTGCGTTTC*CGCAACCTCCATC-3'), 8 STL1-1 GUUCGG Dead inserts two G-C base pairs into the stem adjacent to the 9 STL1-2 CUUCGC Dead tetraloop, in the context of either the wild-type tetraloop or 10 PTL1-1 GUUUCC Viable ++ a lethal point mutant (G16OC) (50% C/50% G at the position 11 PTL1-2 GCUUCC Dead marked with an asterisk). 12 PTL 2C GCUUCA Dead Yeast Methods. Mutant alleles were confirmed by DNA 13 PTL 2U GUUUCA Dead sequencing and introduced into Schizosaccharomyces 14 D4E-1 UUGCGG ... CGCAA Dead pombe strain RM2a, heterozygous for disruption ofthe SRP7 15 D4E-2 UUGCCC ... CGCAA Dead gene (19). Transformation, random spore analysis, and test- 16 D4E-3 UUGG ... CGCAA Viable ing for sensitivity to high or low temperature and/or in- 17 G159C/C164G UUC ... GAA Cold creased osmotic strength were performed as described (13). sensitive + + + To determine generation times, cells were grown in rich 18 G159C/C164A UUC ... AAA Dead medium at 30°C and their density was monitored by counting 19 G159A UUA... CAA Viable +++ in a hemacytometer. 20 G159U UUU ... CAA Viable +++ Construction of the p77 Plasmid Series. Domain IV was 21 C164A UUG... AAA Viable ++ amplified by 25 cycles ofPCR under standard conditions with 22 G159A/C164U UUA ... UAA Viable the two primers D4-PCRI (5'-CTGGCAGTTAGGCCTTG- 23 G159U/C164A UUU ... AAA Viable TAGTACCGA-3'; identical to nucleotides 125-150, except 24 U157G GUG... CAA Viable for the underlined changes to create a Stu I site) and D4- 25 U158C UCG ... CAA Viable PCRII (5'-GCACTGCCCAGGATC-CT*GTAGTGATG-3'; 26 A165G UUG... CGA Viable complementary to positions 172-196 except at the underlined 27 A166C UUG ... CAC Viable ++ nucleotides, which create a BamHI site, and at the position 28 U157G/U158C GCG. .. CAA Viable marked with an asterisk, which restores pairing to the Stu I 29 U157G/A165G GUG ... CGA Viable + site) on pWEC4.2 DNA containing wild-type or mutant 7SL 30 U158C/A166C UCG ... CAC Dead sequences. The products were phenol extracted, digested 31 BP-Comp GCG . CGC Viable with Stu I and BamHI, and ligated into the same sites in pDW19 (a gift of Norman Pace, Indiana University), which critical residues. The striking similarities between the re- has a T7 promoter positioned such that transcription starts at cently determined solution structures of UUCG (11) and the first G of the Stu I site. GAAA (12) tetranucleotide loops prompted us to ask whether In Vitro Transcription. BamHI-digested p77 DNA was they are interchangeable in vivo. Both UUCG substitution transcribed with T7 RNA polymerase according to published mutants in which the flanking residues do not form Watson- procedures (20), and a 10-pmol sample was dephosphorylated Crick base pairs are inviable (Table 1, lines 8 and 9).
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