Satellite Tobacco Necrosis Virus RNA Research Articles

Evidence for Variation in the Optimal Translation Initiation Complex: Plant eIF4B, eIF4F, and eIF(iso)4F Differentially Promote Translation of mRNAs

Eukaryotic initiation factor (eIF) 4B is known to interact with multiple initiation factors, mRNA, rRNA, and poly(A) binding protein (PABP). To gain a better understanding of the function of eIF4B, the two isoforms from Arabidopsis (Arabidopsis thaliana) were expressed and analyzed using biophysical and biochemical methods. Plant eIF4B was found by ultracentrifugation and light scattering analysis to most likely be a monomer with an extended structure. An extended structure would facilitate the multiple interactions of eIF4B with mRNA as well as other initiation factors (eIF4A, eIF4G, PABP, and eIF3). Eight mRNAs, barley (Hordeum vulgare) alpha-amylase mRNA, rabbit beta-hemoglobin mRNA, Arabidopsis heat shock protein 21 (HSP21) mRNA, oat (Avena sativa) globulin, wheat (Triticum aestivum) germin, maize (Zea mays) alcohol dehydrogenase, satellite tobacco necrosis virus RNA, and alfalfa mosaic virus (AMV) 4, were used in wheat germ in vitro translation assays to measure their dependence on eIF4B and eIF4F isoforms. The two Arabidopsis eIF4B isoforms, as well as native and recombinant wheat eIF4B, showed similar responses in the translation assay. AMV RNA 4 and Arabidopsis HSP21 showed only a slight dependence on the presence of eIF4B isoforms, whereas rabbit beta-hemoglobin mRNA and wheat germin mRNA showed modest dependence. Barley alpha-amylase, oat globulin, and satellite tobacco necrosis virus RNA displayed the strongest dependence on eIF4B. These results suggest that eIF4B has some effects on mRNA discrimination during initiation of translation. Barley alpha-amylase, oat globulin, and rabbit beta-hemoglobin mRNA showed the highest activity with eIF4F, whereas Arabidopsis HSP21 and AMV RNA 4 used both eIF4F and eIF(iso)4F equally well. These results suggest that differential or optimal translation of mRNAs may require initiation complexes composed of specific isoforms of initiation factor gene products. Thus, individual mRNAs or classes of mRNAs may respond to the relative abundance of a particular initiation factor(s), which in turn may affect the amount of protein translated. It is likely that optimal multifactor initiation complexes exist that allow for optimal translation of mRNAs under a variety of cellular conditions.

A Novel Interaction of Cap-binding Protein Complexes Eukaryotic Initiation Factor (eIF) 4F and eIF(iso)4F with a Region in the 3′-Untranslated Region of Satellite Tobacco Necrosis Virus

Satellite tobacco necrosis virus (STNV) RNA is naturally uncapped at its 5' end and lacks polyadenylation at its 3' end. Despite lacking these two hallmarks of eukaryotic mRNAs, STNV-1 RNA is translated very efficiently. A approximately 130-nucleotide translational enhancer (TED), located 3' to the termination codon, is necessary for efficient cap-independent translation of STNV-1 RNA. The STNV-1 TED RNA fragment binds to the eukaryotic cap-binding complexes, initiation factor (eIF) 4F and eIF(iso)4F, as measured by nitrocellulose binding and fluorescence titration. STNV-1 TED is a potent inhibitor of in vitro translation when added in trans. This inhibition is reversed by the addition of eIF4F or eIF(iso)4F, and the subunits of eIF4F and eIF(iso)4F cross-link to STNV-1 TED, providing additional evidence that these factors interact directly with STNV-1 TED. Deletion mutagenesis of the STNV-1 TED indicates that a minimal region of approximately 100 nucleotides is necessary to promote cap-independent translation primarily through interaction with the cap binding subunits (eIF4E or eIF(iso)4E) of eIF4F or eIF(iso)4F.

The 5' and 3' extremities of the satellite tobacco necrosis virus translational enhancer domain contribute differentially to stimulation of translation.

The translational enhancer domain (TED) of satellite tobacco necrosis virus (STNV) RNA stimulates translation of uncapped RNAs autonomously. Here we set out to identify the 5' and 3' extremities of TED and features of these sequences with respect to translation. We found that both in wheat germ extract and in tobacco protoplasts, the 5' border is confined to 3 nt. Mutational analysis revealed that the autonomous function of TED is sensitive to 5' flanking sequences. At the 3' end of TED, 23 nt have a cumulative, quantitative effect on translation in wheat germ extract, whereas in tobacco protoplasts, the most 3' 14 nt of these 23 nt do not enhance translation. The 5' and 3' sequence requirements triggered the development of a new secondary structure model. In this model, TED folds into a phylogenetically conserved stem-loop structure in which the essential 5' nucleotides base-pair with the 3' nucleotides that stimulate translation both in vitro and in vivo. Importantly, the 14 3' nucleotides in TED that stimulate translation in the wheat germ extract only do not require the predicted base-pairing in order to function. The discrepancy between in vitro and in vivo sequence requirements thus correlates with potential base-pairing requirements, opening the possibility that TED contains two functional domains.

Functionality of the STNV translational enhancer domain correlates with affinity for two wheat germ factors.

The satellite tobacco necrosis virus RNA is uncapped and requires a 3' translational enhancer domain (TED) for translation. Both in the wheat germ extract and in tobacco, TED stimulates in cis translation of heterologous, uncapped RNAs. In this study we investigated to what extent translation stimulation by TED depends on binding to wheat germ factors. We show that in vitro TED binds at least seven wheat germ proteins. Translation and crosslinking assays, to which TED or TED derivatives with reduced functionality were included as competitor, showed that TED function correlates with binding to a 28 kDa protein (p28). One particular condition of competition revealed that p28 binding is not obligatory for TED function. Under this condition, a 30 kDa protein (p30) binds to TED. Importantly, affinity of p30 correlates with functionality of TED. These results strongly suggest that TED has the capacity to stimulate translation by recruiting the translational machinery either via binding to p28 or via binding to p30.

Features of the autonomous function of the translational enhancer domain of satellite tobacco necrosis virus.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a cap and a poly(A) tail. Translation of STNV RNA in vitro is promoted by a 120-nt translational enhancer domain (TED) in the 3'-untranslated region. TED also stimulates translation of heterologous mRNAs. In this study, we show that TED stimulates translation of a cat mRNA by increasing translation efficiency to the level of capped mRNA. This stimulatory activity is not impaired by translation through TED. TED stimulates translation efficiency from different positions within the mRNA, varying from the 5' end to 940 nt downstream of the coding region. Duplication of TED has an additive effect on translation stimulation only when located at both ends of the mRNA. On dicistronic RNAs, TED stimulates translation of both cistrons to the same extent. These data suggest that TED acts primarily by recruiting the translational machinery to the RNA.

5'- and 3'-sequences of satellite tobacco necrosis virus RNA promoting translation in tobacco.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap and a 3' poly(A) tail. The STNV trailer contains an autonomous translational enhancer domain (TED) that promotes translation in vitro by more than one order of magnitude when combined with the 5'-terminal 173 nt of STNV RNA. We now show that the responsible sequence within the 5' region maps to the first 38 nt of the STNV RNA. Mutational analysis indicated that the primary sequence of the STNV 5' 38 nt and TED is important for translation stimulation in vitro, but did not reveal a role for the complementarity between the two. Translation of chimeric STNV-cat RNAs in tobacco protoplasts showed that TED promotes translation in vivo of RNAs lacking a cap and/or a poly(A) tail. Similar to in vitro, TED-dependent translation in tobacco was stimulated further by the STNV 5' 38 nt.

Expression of Tobacco Necrosis Virus Open Reading Frames 1 and 2 Is Sufficient for the Replication of Satellite Tobacco Necrosis Virus

Tobacco necrosis virus (TNV) is a small icosahedral plant virus which is often associated with satellite viruses. The genomic RNA of TNV contains six open reading frames (ORFs), of which ORFs 1 and 2 are thought to encode the viral polymerase. We demonstrate that tobacco protoplasts transfected with a vector containing TNV ORFs 1 and 2 under the control of the cauliflower mosaic virus 35S promoter, as well as protoplasts derived from transgenic Nicotiana tabacum containing the same gene(s), support replication of satellite tobacco necrosis virus RNA.

Characterization of wheat germ protein synthesis initiation factor eIF-4C and comparison of eIF-4C from wheat germ and rabbit reticulocytes.

Eukaryotic protein synthesis initiation factor (eIF)-4C was purified from wheat germ and the molecular weight was calculated to be approximately 19,000 by SDS-polyacrylamide gel electrophoresis. A similar molecular weight was determined by gel filtration chromatography indicating that wheat germ eIF-4C is functional as a single polypeptide chain. An efficient in vitro translation system dependent upon the addition of eIF-4C was developed. This system was used to determine the concentrations of eIF-4C required for the half-maximal rate of translation of satellite tobacco necrosis virus RNA, alfalfa mosaic virus RNA 4, and barley alpha-amylase mRNA. No significant differences in the concentrations of eIF-4C required for the translation of these mRNAs were observed, although differences were noted for eIF-4A and eIF-4F. This finding suggests that eIF-4C is not involved in the binding of mRNA to 40 S ribosomal subunits. In heterologous assays, rabbit reticulocyte eIF-4C was as active as wheat germ eIF-4C in the wheat germ eIF-4C-dependent system. In addition, wheat germ eIF-4C substituted for rabbit reticulocyte eIF-4C in in vitro assay systems from rabbit reticulocytes. These results indicate that eIF-4C from wheat and rabbit contain conserved functional domains.

The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

The 3' Untranslated Region of Satellite Tobacco Necrosis Virus RNA Stimulates Translation in Vitro

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

The 5' and 3' untranslated regions of satellite tobacco necrosis virus RNA affect translational efficiency and dependence on a 5' cap structure

Satellite tobacco necrosis virus RNA (STNV RNA) is a naturally uncapped viral RNA that contains 1239 nucleotides: 29 in the 5' untranslated region (UTR), 591 in the coding region and 619 in the 3' UTR. Mutations were made in the 5' and 3' UTRs, and the effects of these mutations on translational efficiency and cap independence were measured in an in vitro translation system from wheat germ. Removal of the first 12 nucleotides or 10 changes in the nucleotide sequence of the 5' UTR reduced translational efficiency approximately 3-fold; capping of these 5' mutant mRNAs restored their translational efficiencies. Truncation of the 3' UTR to nucleotide 627 or 700, or deletion of nucleotides 627-737, reduced translational efficiency more than 20-fold; capping of these 3' mutant mRNAs restored their translational efficiencies. These modifications in the 3' UTR increased the concentration of initiation factor 4F required for translation. Chimeric mRNAs were constructed which contained the coding region of rabbit alpha-globin mRNA and either the 5' UTR, 3' UTR, or both the 5' and 3' UTRs of STNV RNA. Both the 5' and 3' UTRs of STNV RNA were necessary to obtain cap-independent translation. These findings indicate that interaction between 5' UTR and the region between nucleotides 627 and 737 in the 3' UTR are required for cap-independent translation.

The absence of a m7G cap on beta-globin mRNA and alfalfa mosaic virus RNA 4 increases the amounts of initiation factor 4F required for translation.

beta-Globin mRNA and alfalfa mosaic virus (AMV) RNA 4, two naturally capped mRNAs, and satellite tobacco necrosis virus (STNV) RNA, a naturally uncapped mRNA, were prepared by in vitro transcription with and without a 5' m7G cap structure (m7G(5')ppp(5')N). The translation of the capped and uncapped forms of these mRNAs was measured in a crude S30 system and a partially purified system from wheat germ. In the S30 system the uncapped forms of beta-globin mRNA and AMV RNA 4 are much less active (greater than or equal to 10%) than their capped forms, whereas the uncapped and capped forms of STNV RNA are equally active. The low activity of uncapped beta-globin mRNA and AMV RNA 4 in the S30 system is due, in part, to inactivation of the uncapped mRNAs in this system. Additional studies, carried out in the partially purified system in which very little inactivation of the mRNAs occurs, show that the uncapped and capped forms of beta-globin mRNA or AMV RNA 4 differ markedly with respect to the amount of eukaryotic initiation factor (eIF)-4F required for translation. For beta-globin mRNA the absence of the 5' cap structure increases the concentration of eIF-4F required for half-maximal translation about 6-fold (from 10 to 60 nM) and for AMV RNA 4 it increases the concentration of eIF-4F about 12-fold (from 5 to 60 nM). The concentrations of eIF-3, eIF-4A, and eIF-4B required for half-maximal translation of the uncapped forms of beta-globin mRNA and AMV RNA 4 are either the same or only slightly higher (1.5- to 2-fold) than the concentrations required for the capped forms. With STNV RNA the concentration of eIF-4F required for half-maximal translation of either uncapped or capped STNV RNA is 3 nM, and the concentrations of eIF-3, eIF-4A, and eIF-4B required for the two forms are also the same. The translation of the capped and uncapped forms of beta-globin mRNA and AMV RNA 4 is inhibited strongly by low concentrations of m7GTP in the partially purified system containing low concentrations of eIF-4F. Under the same conditions, the translation of capped or uncapped STNV RNA is inhibited only slightly by m7GTP. These findings suggest the possibility that the mechanism by which eIF-4F interacts and initiates translation with naturally uncapped mRNAs may not be identical to the mechanism by which eIF-4F interacts and initiates translation of naturally capped mRNAs.

Similarities among plant virus (+) and (−) RNA termini imply a common ancestry with promoters of eukaryotic tRNAs

The 5′ ends of brome mosaic virus (BMV) RNAs contain sequences similar to the consensus internal control region (ICR) of pol III promoters in tRNA genes. Comparison of BMV (+)RNA 5′ termini with BMV (−)RNA termini revealed the presence of two (tandem) repeats of some 30 nucleotides, the more internal containing a region of 73% similarity to the tRNA consensus ICR2 (downstream) region of the ICR. Tandem repeats containing motifs similar to the ICR2 consensus were found at the 5′ termini of (−)RNAs of cucumo-, tobamo-, and tymoviruses whose 3′ (+)RNAs have aminoacylatable tRNA-like structures. Single regions of homology to the BMV(+)RNA 5′ terminus, containing an ICR2-like motif, were detected for several tobravirus RNAs, and for satellite tobacco necrosis virus RNA. The (+)-stranded genomes of these viruses have not been shown to be capable of amino acid esterification. The ICR2 consensus (GGUUCGANUCC) is nearly palindromic, and is contained with the T4 /C loop of tRNAs and viral analogs. Consequently, tRNA promoter-like motifs can be seen at both termini of (+) and (-) RNAs of bromoviruses and other viruses. The presence of ICRI and ICR2-like sequences in BMV genomic 5′ (+)RNAs and the tobamovirus 5′ (−)RNAs may reflect promoter arrangements of primordial genomic RNAs ancestral to both modern plant viruses and eukaryotic tRNAs. Several derivative concepts related to genome evolution are discussed, including the origin of asymmetric strand synthesis of RNAs.

Evidence that the requirements for ATP and wheat germ initiation factors 4A and 4F are affected by a region of satellite tobacco necrosis virus RNA that is 3' to the ribosomal binding site.

A cDNA containing the complete genome of satellite tobacco necrosis virus (STNV) RNA was constructed and cloned into a plasmid vector containing the T7 polymerase promotor. A second clone containing the first 54 nucleotides from the 5' end, which includes the ribosome binding site, was also constructed. RNAs were transcribed from these plasmids (pSTNV1239 and pSTNV54) and tested for their ability to bind to wheat germ 40 S ribosomal subunits in the presence of wheat germ initiation factors eIF-4A, eIF-4F, eIF-4G, eIF-3, eIF-2, Met-tRNA, ATP, and guanosine 5'-(beta, gamma-imino)triphosphate (GMP-PNP). Maximal binding of the STNV RNA transcribed from pSTNV1239 is obtained only in the presence of all the initiation factors and ATP. In contrast, close to maximal binding of STNV RNA transcribed from pSTNV54 is obtained in the absence of eIF-4A, eIF-4F, eIF-4G, and ATP. A series of deletion clones from the 3' end of the STNV cDNA was prepared, and the requirements for binding to 40 S ribosomal subunits were determined. STNV RNAs containing more than 134 nucleotides from the 5' end require eIF-4A, eIF-4F, eIF-4G, and ATP for maximal binding to 40 S ribosomal subunits, whereas STNV RNAs containing 86 nucleotides or less no longer require ATP and these factors. These findings indicate that a region 3' to the initiation codon affects the requirements for eIF-4A, eIF-4F, eIF-4G, and ATP.

Identification of two messenger RNA cap binding proteins in wheat germ. Evidence that the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F are antigenically distinct polypeptides.

Previous work has shown that eukaryotic initiation factor (eIF)-4B from wheat germ is a complex containing two subunits, 80 and 28 kDa, and eIF-4F from wheat germ is a complex containing two subunits, 220 and 26 kDa (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 330-333). Here we show that both the 28-kDa subunit of eIF-4B and the 26-kDa subunit of eIF-4F cross-link to the 5' terminus of capped and oxidized satellite tobacco necrosis virus RNA in the absence of ATP and that the cross-linking of both polypeptides is inhibited by m7GDP. Several lines of evidence indicate that the 28-kDa and the 26-kDa cap binding proteins of eIF-4B and eIF-4F are antigenically distinct polypeptides. Rabbit polyclonal antibodies raised to intact eIF-4B or to the isolated 28-kDa subunit of eIF-4B react strongly with the 28-kDa subunit of eIF-4B on immunoblots, but show only a very weak reaction with the 26-kDa subunit of eIF-4F under the same conditions. In addition, a mouse monoclonal antibody was obtained that reacts strongly with the 26-kDa subunit of eIF-4F but does not react with the 28-kDa subunit of eIF-4B. Evidence is presented also which indicates that the higher molecular weight subunits of eIF-4B and eIF-4F are antigenically distinct. Rabbit polyclonal antibodies raised to intact eIF-4B or the isolated 80-kDa subunit inhibit eIF-4B-dependent polypeptide synthesis but do not inhibit eIF-4F-dependent polypeptide synthesis. Rabbit polyclonal antibodies raised to eIF-4F inhibit eIF-4F-dependent polypeptide synthesis but do not inhibit eIF-4B-dependent polypeptide synthesis.

ATPase activities of wheat germ initiation factors 4A, 4B, and 4F.

Eukaryotic initiation factors (eIF)-4A, -4B, and -4F isolated from wheat germ were tested for their ability to catalyze ATP hydrolysis in the absence and presence of RNA. We find that eIF-4B or eIF-4F alone exhibit ATPase activity in the presence of poly(U), satellite tobacco necrosis virus (STNV) RNA, or globin mRNA but not in the absence of RNA. eIF-4A alone exhibits ATPase activity in the absence of RNA, but this activity is not stimulated by the addition of RNA. eIF-4A does, however, enhance RNA-dependent ATP hydrolysis in the presence of either eIF-4B or eIF-4F. The RNA-dependent ATPase activities of eIF-4B and eIF-4F are additive, not synergistic. At saturating concentrations of eIF-4F, little or no stimulation of ATP hydrolysis is obtained upon the addition of eIF-4B and at saturating concentrations of eIF-4B little or no stimulation is obtained upon the addition of eIF-4F. This observation is in agreement with our previous finding (Lax, S., Fritz, W., Browning, K., and Ravel, J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 330-333) that initiation of polypeptide synthesis is obtained in vitro with either eIF-4F or eIF-4B.

Controlled synthesis of the coat protein of satellite tobacco necrosis virus in Escherichia coli

Chimeric plasmids were constructed such that the cloned complete satellite tobacco necrosis virus (STNV) RNA information came under transcriptional control of the leftward promoter (P L) of bacteriophage λ. The promoter is fully repressed at low temperatures (28°) by the thermolabile repressor product of the λcI857 gene, present in the bacterium on a deficient prophage or as part of another plasmid. Synthesis of the STNV coat protein in Escherichia coli could be initiated by heat induction (42°). These in vivo results confirm that the 5′-untranslated region of STNV RNA, preceding the initiating AUG, provides adequate genetic information for efficient translation in the bacterial system. However, fast degradation of the bacterially synthesized STNV coat protein was observed. The use of a protease-deficient strain reduced this breakdown considerably.

Eucaryotic initiation factor 4B of wheat germ binds to the translation initiation region of a messenger ribonucleic acid.

Purified preparations of eucaryotic initiation factor 4B (eIF4B) from wheat germ bind the monocistronic, uncapped, mRNA satellite tobacco necrosis virus RNA (STNV RNA) in nitrocellulose-mediated binding assays. This reaction is mRNA specific and yields dissociation constants (Kd) in the 10(-7)-10(-8) M range, depending upon the particular enzyme preparation tested. Purified wheat germ eIF4A, in the presence or absence of ATP, does not bind STNV RNA efficiently, but added eIF4A and ATP do enhance the efficiency of the eIF4B-dependent binding of STNV RNA. Wheat germ eIF4B binds the oligonucleotide containing the 5'-terminal 52 nucleotides of STNV RNA (designated 1-52) with the same affinity as intact STNV RNA. This binding affinity is less with the 1-44 oligonucleotide of STNV RNA and does not occur with the 1-33 oligonucleotide of STNV RNA that contains the 5'-terminal untranslated region and the initiator AUG codon at positions 30-32 of this mRNA. Wheat germ eIF4B therefore binds the translation initiation region of STNV RNA, and this binding requires up to 20 nucleotides on the 3' side of the initiator AUG codon of this mRNA. Wheat germ eIF4B also efficiently binds an oligonucleotide containing nucleotides from positions 13-52 in from the 5' terminus of STNV RNA, thereby establishing that the postulated 5'-terminal stem and loop secondary structure of STNV RNA [Leung, D. W., Browning, K. S., Heckman, J. E., RajBhandary, U. L., & Clark, J. M., Jr. (1979) Biochemistry 18, 1361-1366] is not functional or essential for this specific binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Translation of satellite tobacco necrosis virus RNA modified by (not equal to)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is inhibited in a wheat germ cell-free system.

It has been shown that (not equal to)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) modification of rabbit globin mRNA results in inhibition of translational initiation. In order to explore the possibility that modification of the 5' cap structure was responsible for this inhibition, the naturally non-capped mRNA from satellite tobacco necrosis virus (STNV) was reacted with BPDE and translated in a wheat germ cell-free system. The extent of modification was 1.3 and 2.9 BPDE residues/molecule. High performance liquid chromatography of the modified nucleosides from enzymatically hydrolyzed STNV RNA revealed that greater than 90% of the nucleoside adducts were substituted at the exocyclic amino group of guanosine. The translational ability of the lower and higher modified STNV, measured by incorporation of [14C]amino acids into acid-precipitable polypeptides is inhibited by 55% and 63%, respectively. Polyacrylamide gel electrophoretic analyses of the translation products indicate that predominantly full-length coat proteins are synthesized but with the carcinogen-modified STNV the amount is reduced. On the other hand, 80S initiation complex formation is not inhibited as measured by binding of the BPDE-modified STNV to ribosomes and followed by glycerol gradient centrifugation. Under these conditions, aurintricarboxylic acid completely inhibits 80S initiation complex formation in the presence of either modified or native STNV. These results suggest that inhibition of in vitro translation of BPDE-modified STNV, in contrast to that of globin mRNA, is not at the level of initiation complex formation but possibly by premature termination of growing polypeptides.

A new method to characterize the translation initiation sites of messenger ribonucleic acids

RNA can be radiolabeled in vitro with 125I in uridine and cytidine residues by initial mercuration of these pyrimidine residues by the method of Dale et al. [Dale, R. M. K., Martin, E., Livingston, D. C., & Ward, D. C. (1975) Biochemistry 14, 2447-2457] and subsequent electrophilic displacement of the mercury with 125I+ generated by the method of Commerford [Commerford, S. L. (1971) Biochemistry 10, 1993-2000]. These two reactions can be manipulated to produce intact high specific activity 125I-labeled mRNA containing approximately equal specific activity in their 5-[125I]iodouridine and 5-[125I]iodocytidine residues. In vitro radiolabeling of satellite tobacco necrosis virus RNA (STNV RNA) by this procedure yields a 125I-labeled mRNA that is biologically active in ribosome protection analyses in that one obtains the correct translation initiation fragments of the mRNA. Exhaustive digestion of a specific 125I-labeled 32 nucleotide long initiation fragment of STNV RNA using four separate nucleotide-specific digestion (hydrolysis) reactions yields four different populations of 125I-labeled digestion products that can be resolved by two-dimensional fingerprint procedures. Characterization of all these 125I-labeled digestion products followed by overlap nucleotide sequence comparisons of these specific digestion products allows a nucleotide sequence determination of the original 32 nucleotide long translation initiation fragment of this mRNA. This suggests that this overall in vitro procedure can be used to determine the nucleotide sequence of the translation initiation site(s) of any mRNA.