Characterization of the adenylation site in the RNA 3'-terminal phosphate cyclase from Escherichia coli

RNA 3'-terminal phosphate cyclases are a family of evolutionarily conserved enzymes that catalyze ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA. The precise function of cyclases is not known, but they may be responsible for generating or regenerating cyclic phosphate RNA ends required by eukaryotic and prokaryotic RNA ligases. Previous work carried out with human and Escherichia coli enzymes demonstrated that the initial step of the cyclization reaction involves adenylation of the protein. The AMP group is then transferred to the 3'-phosphate in RNA, yielding an RNA-N(3')pp(5')A (N is any nucleoside) intermediate, which finally undergoes cyclization. In this work, by using different protease digestions and mass spectrometry, we assign the site of adenylation in the E. coli cyclase to His-309. This histidine is conserved in all members of the class I subfamily of cyclases identified by phylogenetic analysis. Replacement of His-309 with asparagine or alanine abrogates both enzyme-adenylate formation and cyclization of the 3'-terminal phosphate in a model RNA substrate. The cyclase is the only known protein undergoing adenylation on a histidine residue. Sequences flanking the adenylated histidine in cyclases do not resemble those found in other proteins modified by nucleotidylation.     

Cyclases of the 3'-terminal phosphate in RNA: a new family of RNA processing enzymes conserved in eucarya, bacteria and archaea.

The 2',3'-cyclic phosphate termini are produced, as either intermediates or final products, during RNA cleavage by many different endoribonucleases. Likewise, ribozymes such as hammerheads, hairpins, or the hepatitis delta ribozyme, generate 2',3'-cyclic phosphate ends. Discovery of the RNA 3'-terminal phosphate cyclase has indicated that cyclic phosphate termini in RNA can also be produced by an entirely different mechanism. The RNA 3'-phosphate cyclase converts the 3'-terminal phosphate in RNA into the 2',3'-cyclic phosphodiester in the ATP-dependent reaction which involves formation of the covalent cyclase-AMP and the RNA-N3' pp5' A intermediates. The findings that several eukaryotic and prokaryotic RNA ligases require the 2',3'-cyclic phosphate for the ligation of RNA molecules raised a possibility that the RNA 3'-phosphate cyclase may have an anabolic function in RNA metabolism by generating terminal cyclic groups required for ligation. Recent cloning of a cDNA encoding the human cyclase indicated that genes encoding cyclase-like proteins are conserved among Eucarya, Bacteria, and Archaea. The protein encoded by the Escherichia coli gene was overexpressed and shown to have the RNA 3'-phosphate cyclase activity. This article reviews properties of the human and bacterial cyclases, their mechanism of action and substrate specificity. Possible biological functions of the enzymes are also discussed.     

RtcB is the RNA ligase component of an Escherichia coli RNA repair operon

RNA 2',3'-cyclic phosphate ends play important roles in RNA metabolism as substrates for RNA ligases during tRNA restriction-repair and tRNA splicing. Diverse bacteria from multiple phyla encode a two-component RNA repair cassette, comprising Pnkp (polynucleotide kinase-phosphatase-ligase) and Hen1 (RNA 3'-terminal ribose 2'-O-methyltransferase), that heals and then seals broken tRNAs with 2',3'-cyclic phosphate and 5'-OH ends. The Pnkp-Hen1 repair operon is absent in the majority of bacterial species, thereby raising the prospect that other RNA repair systems might be extant. A candidate component is RNA 3'-phosphate cyclase, a widely distributed enzyme that transforms RNA 3'-monophosphate termini into 2',3'-cyclic phosphates but cannot seal the ends it produces. Escherichia coli RNA cyclase (RtcA) is encoded in a σ(54)-regulated operon with RtcB, a protein of unknown function. Taking a cue from Pnkp-Hen1, we purified E. coli RtcB and tested it for RNA ligase activity. We report that RtcB per se seals broken tRNA-like stem-loop structures with 2',3'-cyclic phosphate and 5'-OH ends to form a splice junction with a 2'-OH, 3',5'-phosphodiester. We speculate that: (i) RtcB might afford bacteria a means to recover from stress-induced RNA damage; and (ii) RtcB homologs might catalyze tRNA repair or splicing reactions in archaea and eukarya.     

Purification of RNA 3'-terminal phosphate cyclase from HeLa cells. Covalent modification of the enzyme with different nucleotides

RNA 3'-terminal phosphate cyclase has been purified about 6000-fold to near homogeneity from HeLa cells. The purified protein is a single polypeptide with an Mr of 38,000-40,000 and a Stokes radius of 2.66 nm. The cyclase shows a pH optimum of 8.0-9.0. In the presence of Mg2+ and ATP this enzyme catalyzes the conversion of a 3'-phosphate group into the cyclic 2',3'-phosphodiester at the 3' end of RNA, through formation of a covalent cyclase-AMP intermediate. GTP, CTP and UTP (but not dATP or ADP) can also function as cofactors in the cyclization reaction, although less efficiently (apparent Km values for ATP and GTP are 6 microM and 200 microM, respectively). Consistent with this, the enzyme can be covalently labelled with the four [alpha-32P]NTPs.     

RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract.

HeLa cell extract contains RNA ligase activity that converts linear polyribonucleotides to covalently closed circles. RNA substrates containing 2',3'-cyclic phosphate and 5'-hydroxyl termini are circularized by formation of a normal 3',5' phosphodiester bond. This activity differs from a previously described wheat germ RNA ligase which circularizes molecules with 2',3'-cyclic and 5' phosphate ends by a 2'-phosphomonester, 3',5'-phosphodiester linkage (Konarska et al., Nature 293, 112-116, 1981; Proc. Natl. Acad. Sci. USA 79, 1474-1478, 1982). The HeLa cell ligase can also utilize molecules with 3'-phosphate ends. However, in this case ligation is preceded by an ATP-dependent conversion of the 3'-terminal phosphate to the 2',3' cyclic form by a novel activity, RNA 3'-terminal phosphate cyclase. Both RNA ligase and RNA 3'-terminal phosphate cyclase activities are also present in extract of Xenopus oocyte nuclei, consistent with a role in RNA processing.     

Enzymatic release of 5'-terminal deoxyribose phosphate residues from damaged DNA in human cells.

Activities that catalyze or promote the release of 5'-terminal deoxyribose phosphate residues from DNA abasic sites previously incised by an AP endonuclease have been identified in soluble extracts of several human cell lines and calf thymus. Such excision of base-free sugar phosphate residues from apurinic/apyrimidinic sites is expected to be obligatory prior to repair by gap filling and ligation. The most efficient excision function is due to a DNA deoxyribophosphodiesterase similar to the protein found in Escherichia coli. The human enzyme has been partially purified and freed from detectable exonuclease activity. This DNA deoxyribophosphodiesterase is a Mg(2+)-requiring hydrolytic enzyme with an apparent molecular mass of approximately 47 kDa and is located in the cell nucleus. By comparison, the major nuclear 5'----3' exonuclease, DNase IV, is unable to catalyze the release of 5'-terminal deoxyribose phosphate residues as free sugar phosphates but can liberate them at a slow rate as part of small oligonucleotides. Nonenzymatic removal of 5'-terminal deoxyribose phosphate from DNA by beta-elimination promoted by polyamines and basic proteins is a very slow mechanism of release compared to enzymatic hydrolysis. We conclude that a DNA deoxyribophosphodiesterase acts at an intermediate stage between an AP endonuclease and a DNA polymerase during DNA repair at apurinic/apyrimidinc sites in mammalian cells, but several alternative routes also exist for the excision of deoxyribose phosphate residues.     

Crystal structure of RNA 3'-terminal phosphate cyclase, a ubiquitous enzyme with unusual topology

BACKGROUND: RNA cyclases are a family of RNA-modifying enzymes that are conserved in eucarya, bacteria and archaea. They catalyze the ATP-dependent conversion of the 3'-phosphate to the 2',3'-cyclic phosphodiester at the end of RNA, in a reaction involving formation of the covalent AMP-cyclase intermediate. These enzymes might be responsible for production of the cyclic phosphate RNA ends that are known to be required by many RNA ligases in both prokaryotes and eukaryotes. RESULTS: The high-resolution structure of the Escherichia coli RNA 3'-terminal phosphate cyclase was determined using multiwavelength anomalous diffraction. Two orthorhombic crystal forms of E. coli cyclase (space group P2(1)2(1)2(1) and P2(1)2(1)2) were used to solve and refine the structure to 2.1 A resolution (R factor 20.4%; R(free) 27.6%). Each molecule of RNA cyclase consists of two domains. The larger domain contains three repeats of a folding unit comprising two parallel alpha helices and a four-stranded beta sheet; this fold was previously identified in translation initiation factor 3 (IF3). The large domain is similar to one of the two domains of 5-enolpyruvylshikimate-3-phosphate synthase and UDP-N-acetylglucosamine enolpyruvyl transferase. The smaller domain uses a similar secondary structure element with different topology, observed in many other proteins such as thioredoxin. CONCLUSIONS: The fold of RNA cyclase consists of known elements connected in a new and unique manner. Although the active site of this enzyme could not be unambiguously assigned, it can be mapped to a region surrounding His309, an adenylate acceptor, in which a number of amino acids are highly conserved in the enzyme from different sources. The structure of E. coli cyclase will be useful for interpretation of structural and mechanistic features of this and other related enzymes.     

U2 small nuclear RNA is a substrate for the CCA-adding enzyme (tRNA nucleotidyltransferase).

The CCA-adding enzyme builds and repairs the 3' terminus of tRNA. Approximately 65% of mature human U2 small nuclear RNA (snRNA) ends in 3'-terminal CCA, as do all mature tRNAs; the other 35% ends in 3' CC or possibly 3' C. The 3'-terminal A of U2 snRNA cannot be encoded because the 3' end of the U2 snRNA coding region is CC/CC, where the slash indicates the last encoded nucleotide. The first detectable U2 snRNA precursor contains 10-16 extra 3' nucleotides that are removed by one or more 3' exonucleases. Thus, if 3' exonuclease activity removes the encoded 3' CC during U2 snRNA maturation, as appears to be the case in vitro, the cell may need to build or rebuild the 3'-terminal A, CA, or CCA of U2 snRNA. We asked whether homologous and heterologous class I and class II CCA-adding enzymes could add 3'-terminal A, CA, or CCA to human U2 snRNA lacking 3'-terminal A, CA, or CCA. The naked U2 snRNAs were good substrates for the human CCA-adding enzyme but were inactive with the Escherichia coli enzyme; activity was also observed on native U2 snRNPs. We suggest that the 3' stem/loop of U2 snRNA resembles a tRNA minihelix, the smallest efficient substrate for class I and II CCA-adding enzymes, and that CCA addition to U2 snRNA may take place in vivo after snRNP assembly has begun.     

Rcl1p, the yeast protein similar to the RNA 3'-phosphate cyclase, associates with U3 snoRNP and is required for 18S rRNA biogenesis

RNA 3'-terminal phosphate cyclases are evolutionarily conserved enzymes catalysing conversion of the 3'-terminal phosphate in RNA to the 2',3'-cyclic phosphodiester. Their biological role remains unknown. The yeast Saccharomyces cerevisiae contains a gene encoding a protein with strong sequence similarity to the characterized cyclases from humans and Escherichia coli. The gene, named RCL1 (for RNA terminal phosphate cyclase like), is essential for growth, and its product, Rcl1p, is localized in the nucleolus. Depletion or inactivation of Rcl1p impairs pre-rRNA processing at sites A(0), A(1) and A(2), and leads to a strong decrease in 18S rRNA and 40S ribosomal subunit levels. Immunoprecipitations indicate that Rcl1p is specifically associated with the U3 snoRNP, although, based on gradient analyses, it is not its structural component. Most of Rcl1p sediments in association with the 70-80S pre-ribosomal particle and a 10S complex of unknown identity. Proteins similar to Rcl1p are encoded in genomes of all eukaryotes investigated and the mouse orthologue complements yeast strains depleted of Rcl1p. Possible functions of Rcl1p in pre-rRNA processing and its relationship to the RNA 3'-phosphate cyclase are discussed.     

The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase

A shortened form of the Tetrahymena intervening sequence (IVS) RNA acts as an enzyme, catalyzing nucleotidyl transfer and hydrolysis reactions with oligo(cytidylic acid) substrates [Zaug, A. J., & Cech, T. R. (1986) Science (Washington, D.C.) 231, 470-475]. These reactions involve phosphodiester substrates. We now show that the same enzyme has activity toward phosphate monoesters. The 3'-phosphate of C5p or C6p is transferred to the 3'-terminal guanosine of the enzyme. The pH dependence of the reaction (optimum at pH 5) indicates that the enzyme has activity toward the dianion and much greater activity toward the monoanion form of the 3'-phosphate of the substrate. Phosphorylation of the enzyme is reversible by C5-OH and other oligo(pyrimidines) such as UCU-OH. Thus, the RNA enzyme acts as a phosphotransferase, transferring the 3'-terminal phosphate of C5p to UCU-OH with multiple turnover. At pH 4 and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganic phosphate. Thus, the enzyme has acid phosphatase activity. The RNA enzyme dephosphorylates oligonucleotide substrates with high sequence specificity, which distinguishes it from known protein enzymes.     

Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC )) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3' terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several mt tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.     

Studies on the sequence of the 3'-terminal region of turnip-yellow-mosaic-virus RNA.

A fragment representing the 3'-terminal 'tRNA-like' region of turnip yellow mosaic (TYM) virus RNA has been purified following incubation of intact TYM virus RNA with Escherichia coli 'RNase P'. This fragment, which is 112+3-nucleotides long has been completely digested with T1 RNase and pancreatic RNase and all the oligonucleotides present in such digests have been sequenced using 32P-end labelling techniques in vitro. The TYM virus RNA fragment is free of modified nucleosides and does not contain a G-U-U-C-R sequence. Using nuclease P1 from Penicillium citrinum, the sequence of 26 nucleotides from the 5' end and 16 nucleotides from the 3' end of this fragment has been deduced. The nucleotide sequence at the 5' end of the TYM virus RNA fragment indicates that this fragment includes the end of the TYM virus coat protein gene.     

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.     

The nucleotide sequence of a precursor to the glycine- and threonine-specific transfer ribonucleic acids of Escherichia coli.

The primary nucleotide sequence of an Escherichia coli tRNA precursor molecule has been determined. This precursor RNA, specified by the transducing phage lambdah80dglyTsuA36 thrT tyrT, accumulates in a mutant strain temperature-sensitive for RNase P activity. The 170-nucleotide precursor RNA is processed by E. coli extracts to form mature tRNA Gly 2 suA36 and tRNA Thr ACU/C. The sequence of the precursor is pG-U-U-C-C-A-G-G-A-U-G-C-G-G-G-C-A-U-C-G-U-A-U-A-A-U-G-G-C-U-A-U-U-A-C-C-U-C-A-G-C-C-U-N-C-U-A-A-G-C-U-G-A-U-G-A-U-G-C-G-G-G-T-psi-C-G-A-U-U-C-C-C-G-C-U-G-C-C-C-G-C-U-C-C-A-A-G-A-U-G-U-G-C-U-G-A-U-A-U-A-G-C-U-C-A-G-D-D-G-G-D-A-G-A-G-C-G-C-A-C-C-C-U-U-G-G-U-mt6A-A-G-G-G-U-G-A-G-m7G-U-C-G-G-C-A-G-T-psi-C-G-A-A-U-C-U-G-C-C-U-A-U-C-A-G-C-A-C-C-A-C-U-UOH(tRNA sequences are italicized). It contains the entire primary nucleotide sequences of tRNA Gly2 suA36 and tRNA Thr ACU/C, including the common 3'-terminal sequence, CCA. Nineteen additional nucleotides are present, with 10 at the 5' end, 3 at the 3' end, and the remaining 6 in the inter-tRNA spacer region. RNase P cleaves the precursor specifically at the 5' ends of the mature tRNA sequences.     

2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3′-phosphate RNA end to a 2′,3′-cyclic phosphate via covalent enzyme-(histidinyl-Nϵ)-AMP and RNA(3′)pp(5′)A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2′-phosphate RNA end in high yield. The rate of 2′-phosphate cyclization by RtcA is five orders of magnitude slower than 3′-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA_3′p and RNA_2′p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5′-OH ends to either 3′-phosphate or 2′,3′-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2′-phosphates, which cannot be spliced by RtcB, to 2′,3′-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ⁵⁴ coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.     

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives

The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue.     

Characterization of 3'hExo, a 3' exonuclease specifically interacting with the 3' end of histone mRNA

The 3' end of mammalian histone mRNAs consisting of a conserved stem-loop and a terminal ACCCA interacts with a recently identified human 3' exonuclease designated 3'hExo. The sequence-specific interaction suggests that 3'hExo may participate in the degradation of histone mRNAs. ERI-1, a Caenorhabditis elegans homologue of 3'hExo, has been implicated in degradation of small interfering RNAs. We introduced a number of mutations to 3'hExo to identify residues required for RNA binding and catalysis. To assure that the introduced mutations specifically target one of these two activities of 3'hExo rather than cause global structural defects, the mutant proteins were tested in parallel for the ability both to bind the stem-loop RNA and to degrade RNA substrates. Our analysis confirms that 3'hExo is a member of the DEDDh family of 3' exonucleases. Specific binding to the RNA requires the SAP domain and two lysines located immediately to its C terminus. 3'hExo binds with the highest affinity to the wild-type 3' end of histone mRNA, and any changes to this sequence reduce efficiency of binding. 3'hExo has only residual, if any, 3' exonuclease activity on DNA substrates and localizes mostly to the cytoplasm, suggesting that in vivo it performs exclusively RNA-specific functions. Efficient degradation of RNA substrates by 3'hExo requires 2' and 3' hydroxyl groups at the last nucleotide. 3'hExo removes 3' overhangs of small interfering RNAs, whereas the double-stranded region is resistant to the enzymatic activity.     

The physiological role of RNase T can be explained by its unusual substrate specificity

Escherichia coli RNase T, the enzyme responsible for the end-turnover of tRNA and for the 3' maturation of 5 S and 23 S rRNAs and many other small, stable RNAs, was examined in detail with respect to its substrate specificity. The enzyme was found to be a single-strand-specific exoribonuclease that acts in the 3' to 5' direction in a non-processive manner. However, although other Escherichia coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3'-hydroxyl group is required for the enzyme to initiate digestion. Studies with RNA homopolymers and a variety of oligoribonucleotides revealed that RNase T displays an unusual base specificity, discriminating against pyrimidine and, particularly, C residues. Although RNase T appears to bind up to 10 nucleotides in its active site, its specificity is defined largely by the last 4 residues. A single 3'-terminal C residue can reduce RNase T action by >100-fold, and 2-terminal C residues essentially stop the enzyme. In vivo, the substrates of RNase T are similar in that they all contain a double-stranded stem followed by a single-stranded 3' overhang; yet, the action of RNase T on these substrates differs. The substrate specificity described here helps to explain why the different substrates yield different products, and why certain RNA molecules are not substrates at all.     

Nucleotide sequence and genome organization of foot-and-mouth disease virus.

A continuous 7802 nucleotide sequence spanning the 94% of foot and mouth disease virus RNA between the 5'-proximal poly(C) tract and the 3'-terminal poly(A) was obtained from cloned cDNA, and the total size of the RNA genome was corrected to 8450 nucleotides. A long open reading frame was identified within this sequence starting about 1300 bases from the 5' end of the RNA genome and extending to a termination codon 92 bases from its polyadenylated 3' end. The protein sequence of 2332 amino acids deduced from this coding sequence was correlated with the 260 K FMDV polyprotein. Its processing sites and twelve mature viral proteins were inferred from protein data, available for some proteins, a predicted cleavage specificity of an FMDV encoded protease for Glu/Gly(Thr, Ser) linkages, and homologies to related proteins from poliovirus. In addition, a short unlinked reading frame of 92 codons has been identified by sequence homology to the polyprotein initiation signal and by in vitro translation studies.