key: cord-0966903-gmgbus9i
authors: Kolla, Venkatadri; Chakravorty, Maharani; Pandey, Bindu; Srinivasula, Srinivasa M; Mukherjee, Annapurna; Litwack, Gerald
title: Synthesis of a bacteriophage MB78 late protein by novel ribosomal frameshifting
date: 2000-08-22
journal: Gene
DOI: 10.1016/s0378-1119(00)00264-x
sha: 45713f9e6085b078e4dc902790cf3e5cdca43151
doc_id: 966903
cord_uid: gmgbus9i

Abstract MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.

allow phages like P22 and 9NA to grow in its presence. MB78 contains a 42 kb linear, double-stranded DNA Bacteriophage MB78 is a virulent phage of Salmonella (molecular weight 28×106 Da), which replicates through typhimurium (Joshi et al., 1982; Srinivasula, 1992) .

concatemer formation, subsequently converted to full-Morphologically, physiologically and serologically, it is length phage DNA through 'headful' packaging mechadifferent from the well-known temperate phage P22 and nism. Like P22, MB78 DNA is circularly permuted and related phages as well as a virulent phage 9NA (Murthy, terminally redundant ( Khan et al., 1991a; Pandey, 1992; 1987) . MB78 cannot multiply in minimal medium con- Srinivasula, 1992 ). taining citrate. The chelating agent EDTA is an effective It is now known that two proteins can be expressed inhibitor of its DNA synthesis, whereas EGTA and from a single open reading frame through 'ribosomal orthophenanthroline have practically no effect on the frameshifting'. If the ribosome shifts during translation, development of the phage ( Verma and Chakravorty, one base in either direction, i.e. towards 3∞ or 5∞ ends, 1987) . MB78 is a dominant phage in that it does not the reading frame will be changed. During the process of 'ribosomal frameshifting', two or more proteins can result, starting from a single initiation codon Abbreviations: bp, base pairs; IPTG, isopropyl b-thiogalactosidase; ( Farabaugh and Vimaladithan, 1998 direction (+1 frame shift) has been described in the k The nucleotide sequence reported in this paper has been deposited yeast retrotransposon TY (Belcourt and Farabaugh, in the EMBL/GenBank database under Accession No. X87092. 1990 ), copia-like elements of Drosophila (Saigo et al., been demonstrated for retroviruses (Jacks et al., 1988;  from bacteriophage MB78 is expressed by ribosomal frameshifting. Vickers and Ecker, 1992) , luteoviruses (Brault and Miller, 1992; Prufer et al., 1992) , the bacterial transposon IS 1 (Sekine et al., 1992) and in potato leafroll virus ( Kujawa et al., 1993) . A −1 frameshifting event often controls the levels of expression of viral reverse tran-2. Materials and methods scriptase relative to viral core proteins in retroviruses. Frameshifting is also known to effect gene expression in 2.1. Bacterial strains coronaviruses and even in a bacterial system (Chamorro et al., 1992) . In all these cases, frameshifting occurs as LT2, a Salmonella strain, was originally obtained the ribosome passes a seven nucleotide sequence 5∞ from Dr. Myron Levine, Department of Human XXXYYYZ 3∞ ( X is A, U or G, Y is A or U, and Z is Genetics, University of Michigan, Ann Arbor, MI. E. coli strain KK2186 was a generous gift from Dr. P. any nucleotide), known as the 'slippery site'. Two of the Berget, then at the Department of Biochemistry and three base pairs between the anticodons of each of the Molecular Biology, University of Texas, Houston, TX. two tRNAs and mRNAs can be maintained after the All other bacterial strains were purchased commercially slip into the −1 reading frame. The slippery sequence from GIBCO-BRL Life Technologies. All the chemicals is not the only determinant of frameshifting; secondary were obtained from Sigma Chemical Company, St. signals are also required (Larsen et al., 1995; Atkinson Louis, USA. et al., 1997) . Secondary signals programmed in the mRNA augment shifting at the slippery sequence to give high levels of frameshifting. These signals, called 2.2. Purification of bacteriophage MB78 and isolation of 'stimulators', are very diverse. For example, the +1 its DNA shift for decoding RF2 (release factor) of E. coli requires two stimulators: one is a UGA terminator at codon 26 Phage stocks were prepared as described earlier flanking the shift site on its 3∞ site ( Weiss et al., 1988) ; ( Kolla and Chakravorty, 2000) . Phage DNA was isothe other is a Shine-Dalgarno sequence located three lated as per the method described by Maniatis et al. nucleotides upstream of the shift site ( Weiss et al., (Sambrook et al., 1989; Kolla and Chakravorty, 2000) . 1988). These two stimulators act independently with substantial activity, but their effects are synergistic.

2.3. Isolation of plasmid DNA Pseudoknots, a tertiary interaction involving base pairing between two regions of unpaired bases, are also Plasmid DNAs were isolated by either alkali lysis involved in frameshifting. The model for the pseudoknot method using standard protocols (Sambrook et al., structure was based on biochemical analysis of the 3∞ 1989; Kolla and Chakravorty, 2000) or by Qiagen and end of turnip yellow mosaic virus ( TYMV ) RNA (Pleij Promega columns according to the manufacturer's et al., 1985; Dumas et al., 1987) . In E. coli, ribosomal instructions. DNA from the gels was extracted using protein S4 represses its own synthesis in addition to the Qiagen columns. synthesis of other ribosomal proteins viz. S11, S13 and L17 by binding to a pseudoknot structure. The structure resembles a 'double pseudoknot' linking a hairpin 2.4. Nested deletions by ExoIII upstream of ribosome binding site with sequences 2-10 codons downstream of the initiation codon ( Tang and The deletions were created primarily as described by Draper, 1989) . Pseudoknots also play a role in the Henikoff (1987) . Briefly, cloned DNA fragment (5structural mimicry of tRNA at the 3∞ termini of plant 10 mg) was digested with two different restriction viral RNAs (Pleij et al., 1985) . One of the most intrienzymes e.g. PstI and BamHI. The enzyme PstI produces guing functions of the pseudoknot structure in framea four-base 3∞ overhang, resistant to ExoIII activity, shifting occurs during the translation of certain retroviral while the enzyme BamHI generates 5∞ protrusion, which mRNAs (Jacks et al., 1988; Kujawa et al., 1993;  is accessible to ExoIII ( Weiss, 1976) . After complete Atkinson et al., 1997) . Mutational analyses in mouse digestion with both the enzymes, the DNA sample was mammary tumor virus (MMTV ) (Chamorro et al., deproteinized by extracting with phenol-chloroform and 1992) and in infectious bronchitis virus (IBV ) (Brierley precipitated with ethanol. The DNA pellet was resuset al., 1992) provide strong evidence for the stimulator pended in 25 ml of 1× ExoIII buffer and the Exonuclease structural element being a pseudoknot. The autoregulatreatment carried out as per the recommendations of tion of gp32 in phage T4 also involves a pseudoknot the manufacturer (Promega). Finally, the deleted frag- (Shamoo et al., 1993) . In this investigation, we provide ments were ligated and used for completing the nucleotide sequence as well as for in vivo expressions. evidence for the first time that one of the two late genes 2.5. In vitro transcription and translation RC5C centrifuge. The supernatant was discarded, and the minicell pellet was suspended in 6 ml of M9 medium. The cell suspension was again layered on to sucrose The coding region for 26 and 28 kDa proteins was amplified by PCR and cloned in bacterial expression gradient as before. Again, two-thirds of the minicell layer was collected into a 15 ml of corex cup, and an vectors, pET21a and pET28a. Recombinant DNAs were in vitro transcribed-translated in the presence of equal volume of M9 was added. The optical density of this minicell suspension was measured at 600 nm in a [35S]-methionine in rabbit reticulocyte lysate with a T7-RNA polymerase-coupled TNT kit (Promega)

Hitachi spectrophotometer to determine the volume in which the minicells would be finally suspended to have according to the manufacturer's recommendations. Briefly, reactions were set up in 50 ml volume in an A 600 =2.0/ml (2×1010 cells/ml ). The cells were finally suspended in M9 minimal medium containing 30% Eppendorf tube containing 25 ml of rabbit reticulocyte lysate, 2 ml of reaction buffer, 1 ml of amino acid mix ( V/V ) glycerol, aliquoted into a number of tubes (200 ml in each) and stored at −80°C. The minicells thus stored minus methionine, 1 ml of RNasin (5U ), 1 mg of template DNA, 4 ml of 35S-methionine (1000 Ci/mmol ) and 1 ml could be used for at least a year. of T7 RNA polymerase (20 U ). The final reaction was made up to 50 ml with sterile distilled water, and the 2.8. Expression of plasmid encoded proteins tubes were incubated at 42°C for 90 min to allow the synthesis of proteins. One to 3 ml samples were applied Purified minicells were labeled with 35S-methionine as described previously (Reeve, 1979; Kolla and on to SDS-polyacrylamide gels after denaturing in a sample buffer by boiling.

Chakravorty, 2000). The frozen minicell suspension (0.1 or 0.2 ml ) was thawed slowly and centrifuged for 3 min in a microfuge. The pellet was suspended in 200 ml of 2.6. Sequencing M9 minimal medium to which 3 ml of 10.5% ( W/V ) Difco methionine assay medium were added and incu-Nucleotide sequencing of EcoRI 'F' fragment was carried out by manual sequencing using Sequenase kit bated at 37°C for 90 min, to complete the translation of bacterial mRNAs in the minicells, received from the ( USB) and also by automated sequencing. Forward and reverse sequencing primers were obtained from mother cell. Then, 25 uCi of 35S-methionine were added and incubated for 60 min at 37°C, followed by incuba-Pharmacia ( Uppsala, Sweden).

tion of 5 min after the addition of 10 ml of unlabeled methionine (1%). The cells were then centrifuged at 2.7. Preparation of minicells 12 000 rpm for 3 min, the cell pellet was washed with 500 ml of 10 mM Tris-HCl, pH 7.6, suspended in 20 ml Minicells were prepared as described by Reeve (1979) . E. coli strain DS410, the minicell producing of the same buffer to which 20 ml of 2× sample buffer were added. The labeled proteins were separated by strain, transformed with desired plasmid was grown overnight to stationary phase in 400 ml of terrific broth SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) , at a constant voltage of 50 V. (Sambrook et al., 1989) in the presence of the relevant antibiotics (ampicillin and tetracycline). The culture was examined under a light microscope to observe the forma-2.9. Fluorography tion of long filamentous cells and minicells. When a reasonable number of minicells were visible under the To detect radio labeled proteins, the gels were fluorographed using water-soluble sodium salicylate (Bonner, microscope, the culture was chilled on ice for 20 min and then centrifuged at 4°C for 5 min at 2,000 rpm 1984). The gel was soaked in methanol, acetic acid, and water (5:1:5) for 60 min, followed by a thorough wash (675×g) in GS3 rotor of Sorvall RC5C centrifuge. The supernatant was transferred to fresh GS3 cups and with water (30 volumes of gel ), then immersed in 1 M sodium salicylate, pH 7.0 for 1 h with mild shaking. centrifuged at 7000 rpm (5,278×g) for 20 min in the same centrifuge. The cell pellet was resuspended in 9 ml Finally, the gel was transferred to Whatman No. 1 sheet, dried under vacuum and subjected to fluorography. of M9 minimal medium, and then 4 ml of the suspension were carefully layered on to 10-30% (w/v) sucrose gradients. The gradients were centrifuged at 4°C for 2.10. Cloning and PCR amplification 18 min at 5000 rpm (4122×g) in the HB4 rotor of the Sorvall RC5C centrifuge.

The presumed coding regions for 26 and 28 kDa proteins were amplified by PCR using the following The top two-thirds of the minicell layer was collected into a 30 ml corex cup using Pasteur pipette. To this, forward and reverse primers with overhanging restriction sites. an equal volume of M9 minimal medium was added, and the suspension was centrifuged again at 10 000 rpm Forward primer: 5∞ CCCGGATCCATGAATCGTT-TTTTACGTTAC 3∞ (11 953×g) for 10 min in the SS34 rotor of the Sorvall Reverse primer: 5∞ CCCGAATTCGGCAGGGTT-AGATTT 3∞

The primers were commercially synthesized by GIBCO-BRL Life Technologies (including the primers designed to create mutations at the 3∞ end of the fragment to avoid the frameshift by overlapping PCR). The primers were also used to identify the presence of inserts in the cloned vectors by PCR screening and to determine the nucleotide sequence. The amplified fragments were digested with appropriate restriction enzymes and cloned in-frame into pET21a and pET28a expression vectors at BamHI and XhoI restriction sites. Restriction enzyme analyses and DNA sequencing confirmed the sequence of all the constructs. Cruz Biotechnology) and resolved by SDS-PAGE (10%) and transferred electrophoretically (100 V constant for 1 h) to Hybond ECL nitrocellulose paper (Amersham were named according to the number of bases deleted; for example, +1518 means that 1518 bases were deleted, Life Science). The paper was blocked overnight in 10% nonfat milk, and incubated in 5% non-fat milk with and +1601 means that 1601 bases were deleted and so on from the full-length construct. To ascertain the sizes horse-radish peroxidase-conjugated T7-antibody (1:10 000, Novagen) for 1 h at room temperature. After of the inserts, the deleted plasmid DNAs were digested with HindIII, located 138 bases away from the EcoRI stringent washing, the filter was developed by chemiluminiscent ECL, as described (Amersham, Arlington site ( Fig. 2) . The sequentially deleted DNAs were further confirmed by dot-blot hybridization (data not pre-Heights, IL). sented ). The deleted fragments were cloned into pUC18, and the deletions were confirmed by DNA sequencing.

In order to determine the expression of various deletion mutants, we performed in vivo minicell expression using 35S-methionine [only a few of the sequentially In order to understand more about the physiology and genetics of the phage MB78, the EcoRI 'F' fragment deleted plasmids are presented here ( Fig. 1) ]. Lane 6 (proteins expressed by vector pUC18) shows two pro-(2.3 kb) of the phage was cloned in pUC18 vector (data not shown), and the expression in minicells was exam-teins, including 30 kDa b-lactamase protein. In lane 1, four major proteins of molecular weights 28, 26, 21 and ined. The EcoRI 'F' fragment of MB78 codes for four proteins of mass 28, 26, 21 and 11 kDa ( Fig. 1, lane 1) . 11 kDa expressed from the cloned EcoRI 'F' fragment could be seen. Lanes 2-5 show the proteins expressed The expression of b-lactamase was not strong in cells carrying the EcoRI 'F' fragment, suggesting that the by different sequentially deleted DNAs. The 21 and 11 kDa proteins could not be seen when 1438 bases presence of a strong promoter (Pandey et al., 1997) in the 'F' fragment is interfering with the expression of the were deleted ( lane 2), suggesting that their promoters and ORFs (open reading frames) reside within 1438 bp b-lactamase gene. To characterize the EcoRI 'F' fragment, sequencing of the fragment was carried out with from the original construct. The expression of 28 and 26 kDa proteins was unaffected even after the deletion a nested set of deletions in the target DNA. The clones of 1518 bp from the original construct ( lane 3). The ( Fig. 3A) . Computation also revealed the presence of a slippery sequence with a possible downstream pseu-present study focuses on the characterization of the 28 and 26 kDa proteins. The clone F-1518 expressed 28 doknot structure ( Fig. 3B) . The slippery sequence present in bacteriophage MB78 resembles that of the turnip and 26 kDa proteins, suggesting that their promoters and ORFs are present in 793 bp (1518-2311) of the EcoRI yellow mosaic virus ( Kujawa et al., 1993) . 'F' fragment. After deletion of 1595 bp, the expression of both proteins was reduced simultaneously ( lane 4), 3.4. Effect of 3∞ truncation on the expression and no expression was detected after the deletion of 1723 bp ( lane 5). These results suggest that the expres-We next examined whether the 28 and 26 kDa proteins are expressed from overlapping open reading sion of these two proteins is driven by a common promoter that resides within 1518-1601 bp and that frames by ribosomal frameshifting. If they are expressed from overlapping open reading frames with the same both the proteins are possibly expressed from overlapping open reading frames.

initiation codon, truncation of the gene from the 3∞ end should yield only a single protein. This part of the gene has an internal HindIII restriction site, located 138 bp 3.3. Nucleotide sequence analyses away from the EcoRI site, at the 3∞ end of the fragment. When this portion is deleted, the coding region will be The nucleotide sequence of the EcoRI 'F' fragment was determined by Sanger's dideoxy chain termination reduced to 555 bp, encoding a protein of approximately 22 kDa. To test this, plasmid +1518 was truncated method (Sanger et al., 1977) using a set of deletion mutants produced by ExoIII (Accession No. X87092).

(+1518/138 H ) and used to transform the minicell producing strain DS410 to observe the expression of Computer analysis of the nucleotide sequence indicated the possibility of encoding four proteins that could be proteins. The expression pattern of the deleted plasmid is presented in Fig. 4 . Deletion of 138 bp from the 3∞ expressed from three ORFs (data not presented ). The ORF for the 28 and 26 kDa proteins starts from 1641 end of the EcoRI 'F' fragment resulted in complete abolition of 26 and 28 kDa proteins, but a major protein, and does not have a stop codon in the 'F' fragment; it appears that the vector stop codon, located adjacent to smaller in size (22 kDa, marked with triangle), was expressed ( lane 4). The deletion of 3∞ end of the gene, EcoRI site, might be used. Analysis of the nucleotide sequence of EcoRI 'F' fragment revealed that expression resulting in the synthesis of a single protein instead of two proteins, supports the frameshift notion. The pres-of 28 and 26 kDa proteins may have occurred through ribosomal frameshifting. We analyzed the sequence for ence of a slippery sequence and a pseudoknot structure downstream to the putative shift site strengthens the the formation of possible secondary structures, necessary for the process of classical ribosomal frameshifting argument. Fig. 3 . Sequence analysis. (A) Probable secondary structure of mRNA derived from 3∞ 138 nucleotides. (B) Probable slippery sequence and downstream pseudoknot structure. The slippery sequence essential for the frameshift is marked in a box, and the bold nucleotides represent the stop codon where mutations were created. Nucleotides involved in the pseudoknot structure are connected.

In order to examine the phenomenon of frameshift further, we next performed coupled in vitro transcription and translation using rabbit reticulocyte lysate. The nucleotide sequence starting from ATG to the end of the fragment was amplified by PCR with forward and reverse primers, as described in Section 2.10. The amplified DNAs were cloned in-frame into bacterial expression vectors, pET21a and pET28a, at BamHI and XhoI sites and named KVM21 and KVM28, respectively. These DNAs were used to synthesize proteins by in vitro transcription and translation (Promega). The results are presented in Fig. 5 ( lanes 1 and 2) . In vitro transcription-translation of the fragment yielded a major protein (90%) of 28 kDa, with he synthesis of a minor protein of apparent molecular mass 26 kDa (arrows). We observed the formation of dimers from 26 and 28 kDa proteins in the absence of reducing agents DTT (data not shown). These results suggest the synthesis of -methionine in rabbit reticulocyte lysate, as described in Section 2. One to three microliters of these samples, as indicated, were applied on to SDS-polyacrylamide gels after denaturing in a sample buffer by boiling. A major translated protein product (28 kDa) and a minor protein are marked with arrows. The position of possible dimers is also marked with an arrow. Lanes 3 and 4 represent the translation of empty pET28a and pET21a vectors, respectively. able volume of 2× sample buffer and subjected to SDS-PAGE fol-Recombinant proteins with C-terminal His.tag lowed by Western blot. The presence of 26 and or 28 kDa proteins (pET21a) and C and N-terminal His.tags (pET28a) was detected using the ECL kit (Amersham). The molecular weight of the proteins is marked with arrows.

were expressed in E. coli BL-21 DE3. Purified proteins were separated on a 12% SDS-polyacrylamide gel and transferred on to a nitrocellulose membrane, and vector (Fig. 6B ). This suggests that frameshift occurs at the C-terminal region, resulting in the appearance of a Western blotting was performed with T7 specific antibody. A single 28 kDa protein was present in cells truncated 26 kDa protein without a C-terminal His.tag in addition to the expression of a full-length 28 kDa expressing C-terminal His.tag in pET21a vector (Fig. 6A) , whereas 26 and 28 kDa proteins were present protein with a C-terminal His.tag. Mutations were created (deletion of three nucleotides) near the putative in cells expressing N and C-terminal His.tags in pET28a slippery sequence by overlapping PCR and were cloned 4. Conclusions into pET28a. Recombinant proteins were expressed and separated as described above. We predicted that this 1. We demonstrated that two late proteins of bacteriophage MB78 could be derived from the same gene as mutation should result in the loss of frameshift. As expected the mutated recombinant constructs did not a result of ribosomal frameshifting. 2. Ribosomal frameshifting has been well established in yield the 26 kDa protein, but only the 28 kDa protein (Fig. 6C, lane 1) . These results further support ribo-E. coli phages T2 (Du et al., 1997) , T4 (Groisman and Engelberg-Kulka, 1995) and T7 (Condron et al., somal frameshifting. 1991; Sipley et al., 1991; Lewis and Matsui, 1996) but not well documented in the case of Salmonella 3.7. Kinetic expression of 28 and 26 kDa proteins bacteriophages except the previously reported observation in the phage P22 ( Uomini and Roth, 1974) . To examine the functions of the 28 and 26 kDa 3. Two cis elements are essential for ribosomal frameproteins, kinetic expression of phage MB78 was pershifting (Jacks et al., 1988; Blinkowa and Walker, formed. The LT2 cells (host) were infected with phage 1990; Tsuchihashi and Kornberg, 1990), but the exact MB78 at a multiplicity of infection (m.o.i.) 10 and pulsemechanism is not clearly understood. It has been labeled with 35S-methionine for 2 min at different times postulated in −1 frameshift that an anti-Shineafter infection. The labeled proteins were subjected to Dalgarno-like sequence, present at 5∞ to the shift site 12.5% SDS-PAGE followed by fluorography. The (Larsen et al., 1994) , pairs with the 16S rRNA of kinetic study demonstrated that the two proteins are the elongating ribosome to make the ribosome pause, late proteins of bacteriophage MB78 ( Fig. 7) . The proresulting in a frameshift. This feature is observed tein pattern of uninfected cells served as a control. The mostly in prokaryotes. expression of 28 and 26 kDa proteins was low at 2 and 4. An aspect of the current study of bacteriophage 5 min after infection, but from 10 min onwards, their MB78 is that more than 35% of the ribosomes appear synthesis increased significantly until cell lysis. It may to be involved in frameshifting. Reports in the literatherefore be assumed that these two proteins are late ture indicate that the proteins synthesized as a result proteins of phage MB78.

of frameshifting are much less numerous, to a maximum of 10-20%. 5. Amino acid sequence analysis (e.g. LC/MS, MALDI-TOF ) of the 28 and 26 kDa two proteins, encoded by the EcoRI 'F' fragment is required to confirm the ribosome frameshift hypothesis from the present study.

Exponentially growing LT2 cells in minimal medium (M9) at 37°C were infected with phage Atkinson

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide centrifugation, washed with 500 ml of medium and finally suspended in 25 ml of M9 medium. Samples collected at different times as indicated minimal site

Programmed ribosomal framewere lysed in an equal volume of 2× sample buffer and applied on to a 12.5% SDS-polyacrylamide gel. Lane 1, uninfected ( UI ) LT2 cells; shifting generates the Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame. Nucleic Acids lanes 2-7, cells infected with phage MB78 after 2, 5, 10, 15, 30 and 45 min, respectively. BRL low-molecular-weight markers are repre-Res

Fluorography for the detection of radioactivity sented in the left lane. The 28 and 26 kDa proteins are marked with two arrows. in gels

Translational frameshifting mediated phage 9NA, a virulent phage of Salmonella typhimurium

Identi-'slippery-sequence' component of a coronavirus ribosomal framefication of a strong promoter of bacteriophage MB78 that lacks shifting signal

Rohde, sequences stimulating frameshifting in the decoding of gene 10 of W., 1992. Ribosomal frameshifting in plants: a novel signal directs bacteriophage T7

Use of minicells for bacteriophage-directed polypepthe simian retrovirus-1 gag-pro frameshift site

3-D graphics modelling of the scriptase-like enzyme in a transposable genetic element in Drosoph-tRNA-like 3∞-end of turnip yellow mosaic virus RNA: structural ila melanogaster

Effect of frameshift-inducing Press, Cold Spring Harbor, NY. mutants of elongation factor 1alpha on programmed +1 frame

Unidirectional digestion with exonuclease III in encoded by IS1: Identification of the site of translational frameshift-DNA sequence analysis

MB78, a specific recognition of an RNA pseudoknot structure

Repesis and gene 10 frameshifting in Escherichia coli showing different lication, maturation and physical mapping of bacteriophage MB78 degrees of ribosomal fidelity

Molecular analysis of bacteriophage MB78

Cloning, sequencing, expression and Ph

Unusual mRNA pseudoknot strucpromoter analysis of a structural protein of bacteriophage MB78. ture is recognized by a protein translational repressor

Translational frameshifting gentural requirements for efficient translational frameshifting in the erates the gamma subunit of DNA polymerase III holoenzyme. synthesis of the putative viral RNA-dependent RNA polymerase Proc

Cleavage of structural proteins during the mutants of bacteriophage P22

thesis is specifically inhibited by the chelating agent EDTA. FEMS rRNA-mRNA base pairing stimulates a programmed -1 ribosomal Microbiol

Enhancement of ribosomal frame

Maldoshifting by oligonucleotides targeted to the HIV gag-pol region

Endonuclease II of Escherichia coli is exonuclease III. 1123-1129

Reading frame switch caused by base-pair formation 70, 2869-2875. between the 3∞ end of 16S rRNA and the mRNA during elongation of protein synthesis in Escherichia coli

Biochemical characterization of the bacterio

The authors are thankful to the Department of Biotechnology, Government of India, the University Grants Commission and the National Institutes of Health (NIH ) (to V.K ) for financial assistance and Dr. Ravikumar Rallapalli for discussion.