RNA-primed DNA Research Articles

Identification of primary initiation sites for DNA replication in the hamster dihydrofolate reductase gene initiation zone.

Mammalian replication origins appear paradoxical. While some studies conclude that initiation occurs bidirectionally from specific loci, others conclude that initiation occurs at many sites distributed throughout large DNA regions. To clarify this issue, the relative number of early replication bubbles was determined at 26 sites in a 110-kb locus containing the dihydrofolate reductase (DHFR)-encoding gene in CHO cells; 19 sites were located within an 11-kb sequence containing ori-beta. The ratio of approximately 0.8-kb nascent DNA strands to nonreplicated DNA at each site was quantified by competitive PCR. Nascent DNA was defined either as DNA that was labeled by incorporation of bromodeoxyuridine in vivo or as RNA-primed DNA that was resistant to lambda-exonuclease. Two primary initiation sites were identified within the 12-kb region, where two-dimensional gel electrophoresis previously detected a high frequency of replication bubbles. A sharp peak of nascent DNA occurred at the ori-beta origin of bidirectional replication where initiation events were 12 times more frequent than at distal sequences. A second peak occurred 5 kb downstream at a previously unrecognized origin (ori-beta'). Thus, the DHFR gene initiation zone contains at least three primary initiation sites (ori-beta, ori-beta', and ori-gamma), suggesting that initiation zones in mammals, like those in fission yeast, consist of multiple replication origins.

A novel specificity for the primer-template pairing requirement in Tetrahymena telomerase.

Telomerase is a specialized reverse transcriptase with a built-in RNA template. Base pairing between the templating domain of telomerase RNA and a telomeric DNA primer is normally a characteristic of elongation of telomeric DNA. Here we demonstrate the mechanism by which Tetrahymena telomerase bypasses a requirement for template-primer pairing in order to add telomeric DNA de novo to completely non-telomeric DNA primers. We show that this reaction initiates by copying the template residue at the 3' boundary of the telomerase RNA template sequence. Unexpectedly, as the RNA template moves through the telomerase catalytic center, the number of required potential Watson-Crick base pairs between RNA template and DNA primer increases from zero to five. We propose that this unprecedented position specificity of a base pairing potential requirement in a polymerase underlies the chromosome healing mechanism of telomerase, and reflects constraints inherent in an internal template.

Role of methionine 184 of human immunodeficiency virus type-1 reverse transcriptase in the polymerase function and fidelity of DNA synthesis.

Methionine 184 of HIV-1 RT is a constituent of the catalytically crucial and highly conserved YXDD motif in the reverse transcriptase class of enzymes. We investigated the role of this residue by substituting it with Ala and Val by site-directed mutagenesis followed by extensive characterization of the two mutant enzymes. The kinetic parameters governing DNA synthesis directed by RNA and DNA templates indicated that both M184A and M184V mutants are catalytically as efficient as the wild type enzyme. Photoaffinity labeling of both the mutant and the wild type enzyme exhibited an identical affinity for RNA-DNA and DNA-DNA template primers. We further demonstrate that M-->V substitution at 184 position significantly increases the fidelity of DNA synthesis while M-->A substitution results in a highly error-prone enzyme without having compromised its efficiency of DNA synthesis. The M184V mutant exhibited a 25-45-fold increase in mismatch selectivity (ratio of k(cat)/K(m) of correct versus incorrect nucleotides) as compared to the WT enzyme. This pattern of error-prone synthesis is also confirmed by examining the abilities of the enzyme-(template-primer) covalent complexes to incorporate correct versus incorrect nucleotide onto the immobilized template-primer. The nature of error-prone synthesis by the M184A mutant shows an increase in both the mismatch synthesis and extension of the mismatched primer termini. Using a three-dimensional molecular model of the ternary complex of HIV-1 RT, template-primer, and dNTP, we observe that the strategic location of M184 may allow it to interact with the sugar moiety of either the primer nucleotide or the dNTP substrate.

The Effects of Cytosine Arabinoside on RNA-primed DNA Synthesis by DNA Polymerase α-Primase

Oligonucleotides containing a specific initiation site for polymerase alpha-primase (pol alpha-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3' terminus of a full-length RNA primer with a 400-fold preference over CMP. The araCMP is elongated efficiently by pol alpha in the primase-coupled reaction. Extension from RNA 3'-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3'-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol alpha is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3' terminus. These results show that the pol alpha-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol alpha and less so from inhibition of primer synthesis by primase.

Enzymatic characterization of human immunodeficiency virus type 1 reverse transcriptase resistant to multiple 2',3'-dideoxynucleoside 5'-triphosphates.

A set of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the polymerase domain of reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), which confers on the virus a reduced sensitivity to multiple therapeutic dideoxynucleosides (ddNs), has been identified. In this study, we defined the biochemical properties of RT with such mutations by using site-directed mutagenesis, overproduction of recombinant RTs, and steady-state kinetic analyses. A single mutation, Q151M, which developed first among the five mutations in patients receiving therapy, most profoundly reduced the sensitivity of RT to multiple ddN 5'-triphosphate (ddNTPs). Addition of other mutations to Q151M further reduced the sensitivity of RT to ddNTPs. RT with the five mutations proved to be resistant by 65-fold to 3'-azido-2',3'-dideoxythymidine 5'-triphosphate (AZTTP), 12-fold to ddCTP, 8.8-fold to ddATP, and 3.3-fold to 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP), compared with wild-type RT (RTwt). Steady-state kinetic studies revealed comparable catalytic efficiency (kcat/Km) of RTs carrying combined mutations as compared with that of RTwt (< 3-fold), although a marked difference was noted in inhibition constants (Ki) (e.g. Ki of a mutant RT carrying the five mutations was 62-fold higher for AZTTP than that of RTwt). Thus, we conclude that the alteration of RT's substrate recognition, caused by these mutations, accounts for the observed multi-ddN resistance of HIV-1. The features of multi-ddNTP-resistant RTs should provide insights into the molecular mechanism of RT discriminating ddNTPs from natural substrates.

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.

Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro.

Complete enzymatic replication of DNA from the simian virus 40 origin has been reconstituted with T antigen and highly purified cellular proteins. DNA polymerase-alpha/primase functions primarily to synthesize RNA-DNA primers for initiation of DNA replication at the origin and for priming each Okazaki fragment. A polymerase switching mechanism requiring replication factor C and the proliferating cell nuclear antigen allows two molecules of DNA polymerase-delta to replicate both strands of the double helix conjointly.

RNA priming coupled with DNA synthesis on natural template by calf thymus DNA polymerase alpha-primase.

A bovine genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by calf thymus DNA polymerase alpha-primase complex. About 7% of the single-stranded DNA clones contained distinct initiation sites consisting of pyrimidine clusters of pyrimidine-rich sequences. The initiation sites were located at or near the 3'-end of the pyrimidine clusters. One of these sequences, containing a 10-mer pyrimidine cluster with major initiation sites, was analyzed in detail. By the successive substitutions of pyrimidines in the cluster with oligodeoxydenylate [(dA)n] in the 5' to 3' direction, the minimum length of the initiation sequence was estimated to be as long as the 7-mer. In contrast, when one or three pyrimidines at the 3'-end of the cluster were replaced with (dA)n, the priming activity was largely lost, indicating that these pyrimidine residues were indispensable for priming. Furthermore, base substitutions of upstream or downstream sequences outside the pyrimidine cluster also decreased the total priming frequencies. Interestingly, the base substitutions inside or outside of the pyrimidine cluster sometimes caused a shift in the major priming sites. These results indicate that the minimum priming unit of the CTPPS1 template for RNA-primed DNA synthesis consists of a pyrimidine cluster (6-mer) with one purine at its 3'-border and that both the 3'-downstream 6-bases and the 5'-upstream 17-bases modulate the priming by enhancing the priming frequency and/or slightly shifting the sites of initiation of primer synthesis. It was also revealed that the lengths of the product RNA primers became shorter as the length of pyrimidine cluster was shortened by substitution with (dA)n. The gel retardation assay further showed that the complex formation between DNA polymerase alpha-primase and the DNA templates was strongly in competition with poly(dC), poly(dG), and poly(dT) but not with poly(dA). Furthermore, template activities as well as the pyrimidine contents of a series of base-substituted DNA correlated well with their affinities to the enzyme, as measured by both gel retardation assay and their Km values for the priming reaction. Apparently, DNA polymerase alpha-primase primarily recognizes the minimum priming unit consisting of a pyrimidine cluster with a purine at the 3'-boundary of purine, but the initiation of primer RNA synthesis can be modified by pyrimidine residues outside of the minimum priming unit.

Primer RNA chain termination induced by 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. A mechanism of DNA synthesis inhibition.

The studies described herein were aimed at defining the mechanism by which 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (FaraATP), the active intracellular metabolite of fludarabine phosphate, inhibits the synthesis of primer RNA and RNA-primed DNA by the polymerase alpha-primase complex. Incubation of the purified DNA polymerase alpha-primase complex with a poly(dT) template, 500 microM ATP, and increasing concentrations of FaraATP from 2.5 to 50 microM resulted in the progressive accumulation of smaller oligoribonucleotides (2-6 nucleotides) at the expense of the full-length products of DNA primase (7-10 nucleotides). Comparison of the kcat/KM values for incorporation of FaraATP and ATP into oligoribonucleotides revealed that DNA primase incorporated FaraATP 30-fold more efficiently than ATP. FaraAMP was present exclusively at the 3'-termini of the growing primer RNA chains, which prevented further elongation of the primers by DNA primase (primer RNA chain termination). At all FaraATP concentrations tested, inhibition of RNA-primed DNA synthesis was accompanied by primer chain termination. In contrast, DNA polymerase alpha added FaraATP onto full-length primer RNAs about 8-fold less efficiently than dATP, and the incorporation of FaraAMP at the 3'-termini of the primers did not prevent further elongation of these primers by DNA polymerase alpha. These results indicate that primer RNA chain termination is the major effect responsible for the inhibition of RNA-primed DNA synthesis by fludarabine phosphate.

RNA-primed complementary-sense DNA synthesis of the geminivirus African cassava mosaic virus.

The plant DNA virus African cassava mosaic virus (ACMV) is believed to replicate by a rolling circle mechanism. To investigate complementary-sense DNA (lagging strand) synthesis, we have analysed the heterogenous form of complementary-sense DNA (H3 DNA) from infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and blot hybridisation. The presence of an RNA moeity is demonstrated by comparison of results for nucleic acids resolved on neutral/alkaline and neutral/formamide gels, suggesting that complementary-sense DNA synthesis on the virus-sense single-stranded DNA template is preceded by the synthesis of an RNA primer. Hybridisation with probes to specific parts of ACMV DNA A genome indicates that synthesis of the putative RNA primer initiates between nucleotides 2581-221, a region that includes intergenic sequences that have been implicated in geminivirus DNA replication and the control of gene expression.

Superoxide dismutase activity regulation by spermine: A new dimesion in spermine biochemistry and sperm development

Amines are a group of highly important compounds of biological importance; they are known promoters of cell growth, can complex with nucleic acids and can stimulate DNA-primed RNA polymerase activity. Spermine, a polyamine abundantly present in the secretions of the male accessory sex organs, has recieved no functional attributes till date. This study had been a pioneer attempt to validate the hypothesis of a metalloenzyme activity modulation by spermine and implicit correlations have been drawn.

T-antigen binding to site I facilitates initiation of SV40 DNA replication but does not affect bidirectionality.

SV40 origin auxiliary sequence 1 (aux-1) encompasses T-antigen (T-ag) binding site I and facilitates origin core (ori-core) activity in whole cells or cell extracts. Aux-1 activity depended completely upon its sequence, orientation and spacing relative to ori-core. Aux-1 activity was lost either by inserting 10 base pairs between aux-1 and ori-core or by placing either orientation of aux-1 on the opposite side of ori-core. Reversing the orientation of aux-1 in its normal position actually inhibited replication. Easily unwound DNA sequences that stimulate yeast or E. coli origins of replication could not replace aux-1. Aux-1 did not affect bidirectional replication. Replication remained bidirectional even when aux-1 was inactivated, and deletion of aux-1 did not affect selection of RNA-primed DNA synthesis initiation sites in the origin region: the transition from discontinuous to continuous DNA synthesis that marks the origin of bidirectional replication occurred at the same nucleotide locations in both wild-type and aux-1 deleted origins. These results support a model for initiation of SV40 DNA replication in which T-ag binding to aux-1 (T-ag binding site I) facilitates the efficiency with which T-ag initiates replication at ori-core (T-ag binding site II) without affecting the mechanism by which initiation of DNA replication occurs.

Human immunodeficiency virus reverse transcriptase. Substrate and inhibitor kinetics with thymidine 5'-triphosphate and 3'-azido-3'-deoxythymidine 5'-triphosphate

3'-Azido-3'-deoxythymidine 5'-triphosphate (AZTTP) was an efficient substrate for the human immunodeficiency virus 1 reverse transcriptase. It was incorporated into both homopolymer and defined sequence DNA-primed RNA templates and DNA-primed DNA templates. The substrate and inhibitor kinetics of both AZTTP and dTTP were dependent on the template-primer and reaction conditions used. dTMP was incorporated into poly(rA).oligo(dT) and into a defined sequence DNA-primed RNA template (when the other three 2'-deoxynucleoside 5'-triphosphates were present) as a conventional substrate, with steady-state Km values of 5-10 microM. The results suggest that the reverse transcriptase was capable of processive DNA polymerization on these DNA-primed RNA templates. In contrast, in the absence of the other three 2'-deoxynucleoside 5'-triphosphates, the time course for incorporation of dTMP into the same defined sequence DNA-primed RNA template was biphasic. A burst of product formation was observed followed by a slow steady-state rate with a Km value of 0.082 microM. AZTMP incorporation into poly(rA).oligo(dT) and into the defined sequence DNA-primed RNA template produced similar biphasic time courses and steady-state Km values. These results were consistent with rate-limiting dissociation of the polymerase.template-primer complex after "forced" termination of polymerization. AZTMP and dTMP were both incorporated into the homopolymer DNA-primed DNA template, poly(dA).oligo(dT), and a defined sequence DNA-primed DNA template as conventional substrates. Their Km values were similar (2-10 microM). The absence of biphasic time courses suggested that dissociation of the DNA-primed DNA templates from the enzyme, after forced termination, was not rate-limiting. This was consistent with a more distributive mode of DNA polymerization. With the defined sequence template-primers and poly(dA).oligo(dT), Ki values for both dTTP and AZTTP were comparable to their Km values. Thus, AZTTP appeared to be a simple competitive substrate-inhibitor with respect to dTTP. AZTTP inhibition of dTMP incorporation into poly(rA).oligo(dT) was linear competitive at low concentrations (0-100 nM) of AZTTP (Ki = 35 nM) but became hyperbolic (decreasing potency) at concentrations of AZTTP above this range. A mechanism for this nonlinear inhibition is discussed.

Discontinuous DNA synthesis by purified mammalian proteins.

Five proteins purified from mouse cells acting together efficiently convert a single-stranded circular DNA template to covalently closed duplex circle by a discontinuous mechanism. DNA polymerase alpha/primase with the assistance of alpha accessory factor covers the single-stranded circle with RNA-primed DNA fragments. Primers are removed by a combination of RNase H-1 and a 5'-exonuclease that was identified by its ability to complete this in vitro system. The 5'-exonuclease is required to remove residual one or two ribonucleotides at the primer/DNA junction that are resistant to RNase H-1. Gap filling is by the DNA polymerase alpha/primase, and DNA ligase I converts the DNA fragments to continuous strand. The concerted action of the five proteins emulates synthesis of the staging strand at the replication fork.

Synthesis and distribution of primer RNA in nuclei of CCRF-CEM leukemia cells

The distribution of primer RNA and RNA-primed nascent DNA in nuclei of CCRF-CEM leukemia cells was examined, and the primer RNA purified from the nuclear matrices of these cells was characterized. RNA-primed nascent DNA was radiolabeled by incubating whole-cell lysates with [alpha-32P]ATP and [3H]dTTP in the presence of approximately physiological concentrations of the remaining ribo- and deoxyribonucleoside triphosphates. The primer RNA was purified by cesium chloride density gradient centrifugation and analyzed by polyacrylamide gel electrophoresis. Nuclear subfractionation studies revealed that at least 94% of the primer RNA and RNA-primed nascent DNA were located within the insoluble matrix fraction of the nucleus. The predominant primer RNA isolated from the nuclear matrix was 8-10 nucleotides in length, and several lines of evidence indicated that this oligoribonucleotide was the functional primer RNA. Essentially all of the matrix primer RNA was covalently linked to the newly replicated DNA as demonstrated by its buoyant density in cesium chloride gradients, phosphate-transfer analysis, and sensitivity to DNase I. Analysis of 32P transfer from [alpha-32P]dTTP revealed a random distribution of ribonucleotides at the 3'-end of the primer RNA. Data obtained from mixing experiments indicated that the association of RNA-primed nascent DNA with the nuclear matrix was not the result of aggregation of these fragments with the nuclear matrix. No significant amount of either primer RNA, RNA-primed nascent DNA, or phosphate transfer was detected in the high-salt-soluble (nonmatrix) fraction of the nucleus, although the nonmatrix fraction contained most of the newly replicated DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Murine DNA polymerase.alpha-primase initiates RNA-primed DNA synthesis preferentially upstream of a 3'-CC(C/A)-5' motif.

We find that the purified murine DNA polymerase.alpha-primase complex exhibits the greatest affinity for DNA templates in which CCC occurs 10 nucleotides downstream of a DNA primase initiation site (Km = 6.6 +/- 0.3 pM). Templates with 3'-CCA-5' at this position are less efficiently utilized (Km = 16 +/- 4 pM). Point mutations that disrupt the 3'-CC(C/A)-5' motif further decrease the affinity for DNA approximately 7-fold (Km = 105 +/- 58 pM). Mutations at the primase start site reduce Vmax 2-fold. Template pyrimidines are required for priming, and initiation with ATP is preferred to initiation with GTP. We conclude that a component of the DNA polymerase.alpha-primase complex recognizes a 3'-CC(C/A)-5' motif in the DNA template, downstream of a primase start site, and that this interaction controls site selection and frequency of initiation by DNA primase.

Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.

Properties of a DNA primase from rice cells

We have shown that rice cells are endowed with a primase which is resistant to α-amanitin and depends on added ATP for its polymerizing activity in a cell-free system in the presence of DNA polymerase I from Escherichia coli.Unlike the animal primase, the bulk (about 60%) of this enzyme is found in the flow through of a phosphocellulose column at 20 mM K-phosphate; the rest is retained by the resin and eluted at 140 mM K-phosphate. Both fractions are free from contaminating α and γ-like DNA polymerase activities and most likely represent a unique enzyme activity.Also at variance with the animal primase, the rice enzyme depends on a γ-like DNA polymerase in a cell-free assay mixture for DNA synthesis initiation when the synthetic oligonucleotide poly(dT) is used as the substrate. However, DNA polymerase α is exclusively required to initiate DNA synthesis when an RNA-primed fd phage DNA is used as the template.This induces us to propose that DNA polymerase α is sufficient to initiate nuclear DNA synthesis in plant cells, as has already been shown in the case of animals cells.

Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein.

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.

A gene cluster in the spinach and pea chloroplast genomes encoding one CF 1 and three CF 0 subunits of the H +-ATP synthase complex and the ribosomal protein S2

The regions of the spinach and pea chloroplast genomes containing the ATP synthase genes atpA, atpF and atpH have been sequenced. The encoded proteins, CF 1α, CF 0I and CF 0III, are well conserved between spinach and pea, and analogous to the α, b and c subunits of the Escherichia coli ATP synthase complex. The atpF gene is split by a single intron, and the exon/intron boundaries have been defined by isolating and sequencing a partial cDNA clone. Two other genes, designated atpI and rps2, located upstream from atpH, have also been sequenced. They encode a 27,000 M r hydrophobic protein analogous to the F 0a subunit of E. coli ATP synthase and a basic protein analogous to the S2 protein of the E. coli 30 S ribosomal subunit. Transcriptional analysis by electron microscopy of RNA-DNA hybrids, Northern blotting and primer extension experiments shows that these genes are transcribed and processed into a complex set of transcripts, with 5′ ends mapping upstream from the rps2, atpI and atpH genes.