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Ribosomal Movement Impeded at a Pseudoknot Required for Frameshifting

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Ribosomal Movement Impeded at a Pseudoknot Required for Frameshifting Proc. Nati. Acad. Sci. USA Vol. 89, pp. 8636-8640, September 1992 Biochemistry Ribosomal movement impeded at a pseudoknot required for frameshifting (doubkl-stded RNA vlrus/Saccharomyces cerevisiae vfrus/trnsaation/yeast) CHIALING Tu, TZY-HWA TZENG, AND JEREMY A. BRUENN* Department of Biological Sciences, State University of New York, Buffalo, NY 14260 Communicated by Edwin D. Kilbourne, June 15, 1992 (receivedfor review April 7, 1992) ABSTRACT Translational frameshifting sometimes occurs backward one base to erroneously read the Gly-Phe codons when ribosomes encounter a "shift" site preceding a region of (T.-H.T., unpublished data). unusual secondary strucure, which in at least three cases is In this work, ribosomes are demonstrated to progress known to be a pseudoknot. We provide evidence that ribosones slowly through the pseudoknot, so that "paused" ribosomes have a decreased rate of movement through a pseudoknot accumulate, apparently flush up against its first stem. Mu- required for fram ing. These paused ribosomes are directly tations that eliminate either stem of the pseudoknot greatly situated over the shift sequence. Ribosomal pausing appears to reduce ribosomal frameshifting and ribosomal pausing at this be necessary but not sufficent for frameshfting. site. Only the secondary structure ofthe RNA is important in this region, not its primary sequence. Ribosomal frameshifting is the shifting ofribosomes from one MATERIALS AND METHODS a mRNA reading frame to another at a specific sequence on Vectors for Detection of FrameshUtng. The pGEM7ZCTU during translation. There are numerous examples ofribosomal family ofvectors for detection of ScV frameshifting has been frameshifting in prokaryotic and eukaryotic systems (1, 2). It described, as have the point mutants and deletion mutants is a commonly used mechanism by which RNA viruses used to define the minimal frameshift region and to show that translationally regulate the level of synthesis of proteins. The the pseudoknot is necessary (12). The locations of altered frequency with which frameshifting occurs may vary from a sequences in the mutants used are described in Table 1. few percent to more than halfofthe time a ribosome traverses In Vitro Transcripts. SP6 transcripts of pGEM7ZCTU and the critical sequence (3). Sequences that induce frameshifting its derivatives (12) were made as described (12, 16, 17) in the often include heptanucleotide runs of purines and/or pyrimi- presence ofm7GpppG to cause capping ofthe resultant RNA. dines, a "slippery" or "shift" sequence (4-6), followed by Single-Stranded DNA. The 396-base-pair (bp) Li cDNA unusual secondary and/or tertiary structure (5-7). Alterna- sequence (bases 1783-2179) in pGEM7ZCTU (12) was first tively, rare codons in the original (zero) reading frame may subcloned to the pGEM7Zf(+) vector (Promega) to create cause frameshifting (8). It has been postulated that ribosomes plasmid pGEM7ZCTU. Isolation of single-stranded DNA slip backward or forward into a new reading frame on a shift from pGEM7ZCTU for heelprinting experiments (see below) sequence when they encounter a region that inhibits their was performed as described (18). The Li minus strand cDNA progression (5). Some retrovirus frameshifting commonly oc- is packaged in this construct. curs just prior to a region capable of forming required sec- Heelprinting. Heelprinting was as described (19) with the ondary structure (5) that may form a pseudoknot (9), although following modifications. A 25-IA translation reaction mixture in human immunodeficiency virus type 1 no such downstream contained 3 ug ofcapped RNA, 1 unit ofRNase inhibitor per structure is necessary (6, 10). A pseudoknotjust downstream p1 (RNasin; Promega), 20 nM amino acid mix, and 17.5 p1 of ofthe shift site has been shown to be essential forframeshifting rabbit reticulocyte lysate (Promega). Following a 25-min in the coronavirus infectious bronchitis virus (7), in the Sac- incubation at 26WC and addition of cycloheximide to a final charomyces cerevisiae virus (ScV) (11, 12), and in the retro- concentration of 1 mM, micrococcal nuclease (final concen- virus mouse mammary tumor virus (6). tration, 16 units/p1; Boehringer Mannheim) was added to In ScV, there are two large open reading frames on the plus digest RNA. The volume of the reaction mixture was strand of the double-stranded RNA genome (L1): cap and adjusted to 200 pl with buffer T (19), and ribosomes were pol. The first, cap, encodes the major viral capsid polypep- collected by pelleting through a 120-p1 cushion of 0.25 M tide, and the second, pol, encodes the viral RNA-dependent sucrose in buffer T at 53,000 rpm, 50C for 2.5 hr in a TLS55 RNA polymerase. The two reading frames overlap and a -1 rotor in a Beckman TL100 ultracentrifuge. The top 240 p1 was frameshift is required for translation ofpol (11-13). A region removed after sedimentation; then proteinase K (200 pg/ml) of 71 nucleotides, including a required (11) shift sequence was added. Ribosome-protected RNA fiagments were puri- (1958-1964, GGGUUUA) and a required (11, 12) pseudoknot fied and redissolved in 10 p1 ofdistilled water. One microliter (1969-2022), is sufficient forframeshifting in yeast in vivo and of ribosome-protected fragments and 0.12 ng of a 5'-labeled in wheat germ translation systems in vitro (12). Frameshift oligonucleotide primer with the Li sequence of bases 1892- sequences behave similarly in homologous and heterologous 1907 were annealed to an Li minus strand cDNA comple- systems, except that efficiencies are higher in some heterol- mentary to bases 1783-2179. The annealing reaction mixture ogous contexts in the homologous system (11, 12). The was heated to 650C for 5 min and then placed at 3rC for 1 hr. protein sequence of a ScV frameshift fusion protein demon- Following annealing, 2.5 units of T4 DNA polymerase and strates a ribosomal slippage similar to the -1 frameshift of dNTPs (final concentration, 0.334 mM) were added to the some retroviruses (4, 5, 14, 15): in this case, a tRNAGlY_ annealing reaction mixture (the polymerase accessory pro- tRNAI-eU pair, in the P and A sites, respectively, slips teins gp44/62 and gp45 were omitted from the primer exten- sion reaction because we found them unnecessary). After a The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: ScV, Saccharomyces cerevisiae virus. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 8636 Downloaded by guest on September 25, 2021 Biochemistry: Tu et al. Proc. Natl. Acad. Sci. USA 89 (1992) 8637 Table 1. Correlation between frameshifting and 12 kDa and a frameshift fusion product of 27 kDa result. The ribosomal pausing frameshifting efficiency measured in this manner is about 3% Mutant Bases altered Stem altered FS*, % WT Heelprint in wheat germ extracts (12) and 2.5-5% in the rabbit reticu- locyte lysate system used for the current experiments (21). ml 1969-1973 1 10-15 - Strategy Used for Detecftng "Pausing." We used the "heel- m2 1984-1991 2 10-15 - printing" technique (19, 22) to locate regions protected from m3 2000-2004 1 10-15 - nuclease digestion by ribosomes translating the frameshift m4 2015-2022 2 10-15 - region. This technique locates the 5' extent of regions pro- m5t 1969-1973 tected from micrococcal nuclease digestion by steric hin- 2000-2004 1 40 + drance of the nuclease by proteins bound to RNA (in this m6t 1984-1991 case, ribosomes). SP6 transcripts were incubated in rabbit 2015-2022 2 150 + reticulocyte lysates and then treated with micrococcal nu- d3 2021-2022 clease. The protected RNA fragments were isolated as com- deleted 2 ND + plexes with ribosomes by centrifugation through a sucrose d9 2013-2022 cushion followed by phenol extraction and ethanol precipi- deleted 2 ND tation. The resultant protected fragments were hybridized to The presence (+) or absence (-) of the normal heelprint (ribo- single-stranded DNA with the sequence of the minus strand somal pausing) is indicated for each ofthe mutants tested, along with from the appropriate region of Li (complementary to bases a briefdescription ofeach and the extent offrameshifting in vitro with 1783-2179). Primer extension was accomplished by T4 DNA each. ND, not detectable. *Percent of wild-type frameshifting. polymerase primed with a 5'-end-labeled oligonucleotide tMutant m5 has the changes of ml and m3; mutant m6 has the with the sequence of bases 1892-1907 of the Li plus strand. changes of m2 and m4. The resultant labeled extension products were separated on an 8% polyacrylamide sequencing gel in parallel with the 15-min incubation at 370C, the primer extension products Sanger sequencing reactions generated with the same primer. were purified, precipitated, and redissolved in 10 t4 of Because the RNA-DNA hybrid halts the progression of the sequencing sample buffer (20). Five microliters of extension T4 DNA polymerase, which lacks a 5' exonuclease activity products was denatured and fractionated on each lane of an (23), the length of the extension product reflects the position 8% polyacrylamide sequencing gel. of the 5' end of the protected fragment. This then represents the position of the "heel" of the ribosome protecting the RESULTS RNA from digestion by micrococcal nuclease. The result is Strategy Used for Detecting Frameshifting. We have con- shown as Fig. 1. The protected fragments cause two strong structed a T4 DNA polymerase terminations at bases 1946 and 1949, number of vectors in which the ScV frameshift just prior to the slippery site, beginning at base 1958. We will sequence can direct frameshifting in heterologous contexts, refer to this as the normal heelprint. in vitro and in vivo (12). We examined ribosomal pausing in Ribosomal Pausing Is Correlated with Frameshifting. To pGEM7ZCTU, containing a 396-bp fragment (bases 1783- determine if the strong polymerase termination products 2179) including the ScV shift site (1958-1964) and pseudoknot were correlated with the presence of the pseudoknot, we (1969-2022).
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