MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

MITOCHONDRIAL DNA STRUCTUREAND FUNCTION Carlos T. Moraes, 1 Sarika Srivastava, Ilias Kirkinezos, Jose Oca-Cossio, Corina vanWaveren, Markus Woischnic...
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MITOCHONDRIAL DNA STRUCTUREAND FUNCTION

Carlos T. Moraes, 1 Sarika Srivastava, Ilias Kirkinezos, Jose Oca-Cossio, Corina vanWaveren, Markus Woischnick, and Francisca Diaz Department of Neurology University of Miami School of Medicine Miami, Florida 33136

I. Mammalian Mitochondrial Genomes II. The Human mtDNA III. Structure of the Human mtDNA D-Loop Region IV. Mitochondrial DNA Replication V. Initiation of L-Strand DNA Replication VI. Alternative Mode of mtDNA Replication VII. General Features of Factors Associated with mtDNA Replication A. DNA Polymerase y B. Mitochondrial Single-Strand Binding Protein VIII. Regulation of mtDNA Replication IX. Mitoehondrial Transcription A. Transcription Initiation B. Transcription Elongation and Termination C. Posttranscriptional Modifications X. Translation of Mitochondrial Transcripts XI. Concluding Remarks References

h Mammalian Mitochondrial Genomes

I n 1963, D N A was first d e t e c t e d w i t h i n m i t o c h o n d r i a (N. M. K. Nass a n d S. Nass, 1963). I n t h e n e x t 30 years, t h e c o m p l e t e m i t o c h o n d r i a l D N A ( m t D N A ) s e q u e n c e [ a p p r o x i m a t e l y 17,000 b a s e p a i r s ( b p ) ] was d e t e r m i n e d in m o r e t h a n a d o z e n species, i n c l u d i n g h u m a n s ( A n d e r s o n et al., 1981). M o s t v e r t e b r a t e cells in c u l t u r e a p p e a r to have a p p r o x i m a t e l y 1 0 0 0 - 5 0 0 0 m o l e c u l e s o f t h e c i r c u l a r m i t o c h o n d r i a l g e n o m e ( B o g e n h a g e n a n d Clayton, 1974; S h m o o k l e r Reis a n d G o l d s t e i n , 1983). T h e m t D N A localizes to t h e m i t o c h o n d r i a l m a t r i x a n d s e e m s to b e a s s o c i a t e d with p r o t e i n s a n d l i p i d s ( H i l l a r et al., 1979). I n yeast, t h e l a r g e r [ ~ 8 0 l d l o b a s e s ( k b ) ] m i t o c h o n d r i a l g e n o m e s a r e o r g a n i z e d in 1 0 - 2 0 d i s t i n c t n u c l e o i d s (i.e., p r o t e i n - D N A 1To whom correspondence should be addressed. INTERNATIONALREVIEWOF NEUROBIOLOGY,VOL53

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Copyright2002,ElsevierScience(USA). Allrightsreserved. 0074-7742/02$35.00

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FIG. 1. Structure of the human mitochondrial DNA. Panel A depicts the 16,569-bp human mtDNA showing 13 protein coding genes as well as 2 rRNA- and 22 tRNA-coding genes. Genes coding for subunits of complex I (ND1-ND6), complex III (Cyt b), complex IV (COX ICOX III), and complex V (A8 and A6) are shown by different hatches. The insert in panel A illustrates the mechanisms associated with mtDNA replication and transcription, including the approximately binding sites for the mitochondrial RNA polymerase, the mitochondrial

MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

complexes), which are spherical or ovoid, measuring 0.3-0.6/z in diameter. Nucleoids contain between 3 and 4 mitochondrial genomes and as many as 20 different polypeptides (Miyakawa et al., 1987; Kaufman et al., 2000). It is unclear if animal mtDNA is also organized as DNA-protein complexes (nucleoids), although this possibility has been suggested as a system to maintain genetic stability (Jacobs et al., 2000). One of the most striking differences between the yeast and the animal systems can be observed during development of animal cells. Mitochondrial DNA copy number seems to be strictly controlled during development (Piko and Taylor, 1987; Lefai et al., 2000b), and specific mechanism may have evolved because of these needs. The recent identification of a mitochondrial helicase termed Twinkle, which shows a punctate localization compatible with a nucleoid structure, gives support for this model (Spelbrink et al., 2001). Electron microscopy analyses showed that mammalian mtDNA can be arranged as unicircular monomers, but also as unicircular dimers or catenated forms (Clayton, 1982). These early studies also showed what has been termed the displacement loop or "D-loop" as a separation of strands in a specific region of the mtDNA. It is now known that most sequences associated with initiation of mtDNA replication or transcription are in the proximity of the D-loop region (Clayton, 1982). Both transcription and replication of one strand and transcription of the complementary strand initiate in the proximity of the Dqoop. This l123-bp stretch of DNA is often in a singlestranded configuration, and contains sites for DNA-binding proteins that control mtDNA replication and transcription. Mutations in this region have been observed to accumulate during aging (Michikawa et al., 1999; Wang et al., 9001), but it is still unclear if these alterations affect mtDNA replication or gene expression.

II. The Human mtDNA

The h u m a n mtDNA is representative of mammalian mitochondrial genomes. It is a 16,569-bp, double-stranded, circular molecule encoding 13 polypeptides (Fig. 1). All mtDNA-encoded polypeptides are members of the oxidative phosphorylation complexes (OXPHOS). These include seven

transcription factor mtTFA, the RNA processing enzyme RNAse MRP, and the transcription termination factor mTERF. The origins of replication for the H- and L- (OH and OL) strands a r e also shown. Panel B shows the structure of the regulatory D-loop region in more detail, including the approximate position of the conserved sequence boxes believed to play a role in replication and RNA primer processing. It also shows the location of the two hypervariable regions (HSV1 and HSV2) commonly used for evolutionary studies.

MORAESet al. subunits of complex I, one subunit of complex III, three subunits of complex IV, and two of complex V. Besides protein coding genes, mtDNA also codes for 22 transfer RNAs (tRNAs) and two ribosomal RNAs (12S and 16S rRNAs). The expression and maintenance of mtDNA depends on a large n u m b e r of nuclear-coded factors that are synthesized in the cytosolic ribosomes as precursor polypeptides and imported into the mitochondria via specialized import pores (Attardi and Schatz, 1988). Although the catalytic subunits of the O X P H O S system are e n c o d e d by the mtDNA, these enzyme complexes also contain a large n u m b e r of nuclear-coded subunits that are necessary for their function. The asymmetric distribution of guanine and cytosine permits separation of mtDNA into "heavy" (H-strand) and "light" (L-strand) strands in alkaline density gradient centrifugation. The rRNAs, all but one polypeptide, and 14 o f the 22 tRNAs are e n c o d e d in the heavy-strand genes. In contrast to Saccharomyces cerevisiae mtDNA, vertebrate mtDNA are devoid of introns (Anderson et al., 1981). There are very few n o n c o d i n g intergenic regions, with the exception of the regulatory region containing the promoters and origin of heavy-strand replication. The genetic information is so condensed that there is an overlap in some coding sequences, and termination codons can be generated by the addition of adenines to the transcript during polyadenylation ofmRNAs (Anderson et al., 1981). The genetic code o f vertebrates' mtDNA differs from the nuclear-cytoplasmic code. Instead of being a termination codon, TGA codes for tryptophan in vertebrate's TABLE I MAMMALIANMITOCHONDRIALGENETICCODEa UUU Phe UUC Phe UUA Leu UUG Leu CUU Leu CUC Leu CUA Leu CUG Leu AUU Ile AUC Ile AUA Met(Ile) AUG Met GUU Val GUC Val GUA Val GUG Val

UCU Ser UCC Ser UCA Ser UCG Ser CCU Pro CCC Pro CCA Pro CCG Pro ACU Thr ACC Thr ACA Thr ACG Thr GCU Ala GCC Ala ~ Ala GCG Ala

UAU Tyr UAC Tyr UAA Ter UAG Ter CAU His CAC His CAA Gln CAG Gln AAU Asn AAC Asn AAA Lys AAG Lys GAU Asp GAC Asp GAA Glu GAG Glu

UGU Cys UGC Cys UGA Trp(Ter) UGG Trp CGU Arg CGC Arg CGA Arg CGG Arg AGU Ser AGC Ser AGA Ter(Arg) AGG Ter(Arg) GGU Gly GGC Gly GGA Gly GGG Gly

aAminoacids in parentheses correspond to universal genetic code.

MITOCHONDRIALDNASTRUCTUREAND FUNCTION mitochondria. ATA codes for methionine in mitochondria but isoleucine in the cytosol. Finally, AGA or AGG in mitochondria code for a stop codon instead of arginine (Table I) (Anderson et al., 1981).

IIh Slructure of the Human mtDNA D-Loop Region

Comparison of the nucleotide sequences of mammals' mtDNA revealed some degree of conservation in the promoter regions as well as in three other regions (termed Conserved Sequence Blocks, or CSB I, CSB II, and CSB III) (Walberg and Clayton, 1981). These sequences are conserved in the D-loop regions of many vertebrates, suggesting important roles for these motifs. The CSBs are hypothesized to be involved in some aspect of mtDNA replication because they are located in the D-loop region, and in the case of CSB I, almost always near the initiation site for H-strand DNA synthesis. However, the absence of certain CSBs in some vertebrates suggests that either the function of these elements can be obviated by specific D-loop region configurations or that other novel nucleotide sequences (or protein factors) can provide the same function in these organisms. The majority of the D-loop region contain noncoding sequences and include hypervariable regions (Greenberg et al., 1983). Although the overall rate of mutations in these hypervariable regions are significantly higher than in the rest of mtDNA (Greenberg et al., 1983), some nucleotide positions seem to be hot spots for changes (Stoneking, 2000). The two hypervariable segments (HV1 and HV2; positions 16024-16383 and 57-372, respectively) have been very useful in studying evolution of eukaryotes (Lang et al., 1999), and more specifically, of human populations (Jorde et al., 2000).

IV. Mitochondrial DNA Replication

In most cases, mtDNA replication in mammals is an asynchronous process, beginning at the origin of the H-strand replication (OH) and proceeding around two thirds of the mitochondrial genome, until the origin of the L-strand replication (OL) (Fig. 1) is forced into a single-strand configuration by the extending daughter H-strand. At this point, the displaced H-strand starts to be copied into the daughter L-strand. The precise mapping of RNA and DNA species in the D-loop region provided evidence that RNA derived from the L-strand promoter (LSP) serves as a primer for H-strand DNA replication (Chang and Clayton, 1985; Chang et al., 1985). There is also

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evidence suggesting that the CSBs are involved in formation of a properly configured RNA primer. This RNA synthesized from the L-strand promoter (7S RNA) is correctly processed by a mitochondrial RNA processing (MRP, see below) activity. The existence of an RNA-DNA hybrid downstream of human LSP has been demonstrated (Xu and Clayton, 1996). The human hybrid is confined to a specific region of the origin (spanning CSB II and CSB I), and hybrid formation is virtually abolished by mutations in the upstream CSB III element. A detailed study of the mammalian RNA-DNA hybrid using mouse OH revealed an unusual structure, where a molecule containing an extremely stable R-loop consisting of two DNA strands and one RNA strand (with one of the DNA strands displaced by the hybridized RNA molecule) (Lee and Clayton, 1996). Because this was also observed using a plasmid construct containing the isolated OH region, the nucleic acid sequence of mouse OH appears to contain all of the information required for formation of a stable RNA-DNA hybrid. To provide an appropriate primer for replication, a site-specific mitochondrial RNA processing endoribonuclease (RNase MRP) processes 7S RNA substrates at sites that match some of the RNA-DNA transition sites (i.e., potential DNA replication priming sites) that have been mapped at the H-strand origin in vivo (Dairaghi and Clayton, 1993). The MRP enzyme contains, in addition to protein components, an RNA essential for activity (Chang and Clayton, 1987; Chang and Clayton, 1989). The pattern of RNA cleavage by RNase MRP is consistent with a role for the enzyme in providing primers for mtDNA replication with the substrate being probably the triple-stranded RNA-DNA hybrid, rather than single-stranded RNA. These cleavages seem to be dependent on the presence of CSB I, suggesting that RNase MRP is necessary for the processing that produces the RNA primers in mammalian mitochondria. These findings also implicate the RNA-DNA hybrid as the substrate for the RNA processing that leads to formation of the primers for H-strand replication. RNase MRP activity is also found in the cell nucleus (Chang and Clayton, 1987; Gold et al., 1989). Several lines of evidence demonstrated that nuclear RNase MRP is involved in late stages of 5.8S rRNA processing in the nucleus (Lygerou et al., 1996). However, a small amount ofMRP RNA has been localized to the mitochondrion in mouse cells (Li et al., 1994) and Xenopus laevis oocytes (Davis et al., 1995) by in situ hybridization. In addition, mutations in the genes encoding the RNA component of Saccharomyces cerevisiae and S. pombe RNase MRP RNA have been isolated that result in a mitochondrial phenotype (Paluh and Clayton, 1996). Taken together, these observations suggest the existence of a larger pool of RNAse MRP in the cell nucleus that is responsible for rRNA processing, and a smaller pool in mitochondria that appears to be involved in mitochondrial RNA primer processing.

MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

V. Initiation of L-StrondDNA Replication

In vertebrates, the origin of light-strand replication (OL) is located a large distance from OH on the mtDNA molecule (Figure 1). Initiation of L-strand DNA replication has been studied in mammals, where it occurs within a small [30 base pair(bp)] noncoding region that is flanked by tRNA genes. The DNA sequence in this region has the potential to assume a stable stem-loop structure (Tapper and Clayton, 1981) that is thought to form after the replication fork from initiation of H-strand synthesis passes OL and exposes the parental H-strand in this region as a single strand. Most of our understanding of OL function comes from studies that utilized an in vitro replication system for the h u m a n OL. These studies showed that OL is capable of initiating L-strand DNA synthesis at sites that match those mapped from nucleic acids isolated from mitochondria in vivo (Wong and Clayton, 1985). Initiation of L-strand DNA synthesis requires a DNA primase responsible for generating short RNA molecules with 5t-ends that map to the T-rich portion of the loop in the predicted OL stem-loop structure. In most vertebrates, a noncoding region with conserved predicted secondary structure is found within the sequence in the mtDNA molecule that encodes a cluster of tRNAS for the amino acids Trp, Ala, Asn, Cys, and Tyr (Anderson et al., 1981).

Vh Alternative Mode of mtDNA Replication

Holt and colleagues (2000) proposed that the mammalian mitochondrial genome has two modes of replication. The first one, described above (Clayton, 1982), involves the asymmetric replication of the leading and lagging strands. The second one, based on the observation of replication intermediates in two-dimensional (2D) gels suggested that replication, in a certain number of mtDNA molecules, involves coupled leading- and lagging-strand synthesis. Interestingly, they found a higher percentage of the latter mechanism in ceils that were transiently depleted of their mtDNA. Although there are questions on whether these observations actually reflect alternative modes of mtDNA replication, different modes could have an active role in controlling mtDNA copy number, as specific factors may be involved in different modes of replication. As a possible control mechanism, factors necessary for the coupled leading- and lagging-strand synthesis may be limiting, and once mtDNA levels are close to normal they can no longer participate in the replication of most molecules, thereby decreasing the overall mtDNA replication in the cell.

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VII. General Features of Factors Associated with mlDNA Replication

Many factors involved in mammalian mtDNA replication have been characterized. These include subunits A (catalytic, also referred as subunit 0~) and B (accessory, also known as subunit t ) of DNA polymerase y, mitochondrial RNA polymerase, mitochondrial single-stranded binding protein (mtSSB), mitochondrial transcription factor A (mtTFA), and RNA processing enzymes (reviewed in Lecrenier and Foury, 2000; Shadel and Clayton, 1997). A mitochondrial DNA ligase, apparently related to the nuclear DNA ligase III, that is likely to participate in the resolution of replicated strands also has been characterized (Pinz and Bogenhagen, 1998; Lakshmipathy and Campbell, 1999). Even though the primary functions of these factors are understood, their role in the regulation of mtDNA copy number is not. In addition, mitochondrial helicases (Spelbrink et al., 2001) and topoisomerases (Topcu and Castora, 1995) are also likely to have a role in this process. Experimental evidence also exists suggesting that the cell nucleus may exert a negative control on the mitochondrial genome through some short-lived nuclear substance(s) (Rinaldi et al., 1979).

A. DNA POLYIVIERASEF Overexpression of the catalytic subunit (subunit A) of the mtDNA polymerase F in cultured insect or human cells did not alter mtDNA levels (Lefai et al., 2000a; Spelbrink et al., 2000). However, in transgenic flies overexpressing pol v-A, the number of mitochondrial genomes was reduced drastically, indicating that although cells can tolerate a variable amount of the pol y catalytic subunit under some conditions, the levels of subunit A could be critical in the context of the whole organism (Lefai et al., 2000b). Flies with mutations in pol F-A show problems with the visual system and altered behavior in the wandering stage, both of which seemed to be a consequence of defects in locomotion (Iyengar et al., 1999). The expression of the accessory subunit of pol F (subunit B) seems to correlate better with mtDNA replication activity. The steady-state level ofpol F-B mRNA increases during the first hours of development, reaching its maximum value at the start of mtDNA replication in Drosophila embryos. This pattern of expression was not observed with pol F-A mRNA (Lefai et al., 2000b). A potential link between nuclear and mitochondrial DNA replication also has been described in Drosophila. The pol y-B promoter contains a DNA replication-related site (DRE), previously identified in genes involved in nuclear DNA replication, which is essential for its transcription, suggesting a common regulatory

MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

mechanism controlling nuclear and mitochondrial DNA replication (Lefai et aL, 2000b). Recendy, a mutation in the DNA pol F subunit Awas associated with multiple mtDNA deletions in patients with progressive external ophthalmoplegia (Goethem et al., 2001).

B. MITOCHONDRIAL SINGLE-STRAND BINDING PROTEIN

The mtSSB also has an important role in mtDNA replication. The rate of DNA synthesis by Drosophila DNA polymerase y was increased nearly 40-fold upon addition of mtSSB. The stimulation of both 5'-3' DNA polymerase and 3'-5' exonuclease activities of Drosophila pol F by mtSSB results from increased primer recognition, binding, and rate of initiation (Farr et al., 1999). Similar to the pol y-B promoter, putative transcription factor binding sites clustered within the promoter region of the mtSSB gene include two Drosophila DREs. Deletion and base substitution mutagenesis of the DRE sites demonstrated that they are required for efficient promoter activity, and gel electrophoretic mobility shift analyses showed that DRE binding factor (DREF) binds to these sites (Ruiz De Mena et al., 2000). The link between mitochondriai and nuclear DNA replication is probably very complex and regulated by additional factors as mRNA levels for mtSSB varies independently of cell proliferating activity (Ruiz De Mena et aL, 2000). Flies with a disruption in the mtSSB show a marked mtDNA depletion, defective mitochondrial respiration, and a "low-power" phenotype, similar to the one observed in mutants ofpol F-A (Maier et aL, 2001).

VIII. Regulation of mtDNA Replication The available information on the regulation of mtDNA replication factors in adult animal cells is compatible with the concept that the levels of these factors do not increase significantly when mtDNA levels decrease. Schultz et al. (1998) found that the DNA pol y subunit A is expressed at similar levels in different tissues and does not seem to be regulated by physiological changes. Davis et al. (1996) showed that DNA pol y-A transcripts and protein levels in human cells devoid of mtDNA were comparable with those of controls. Larsson et al. (1994) and Moraes et al. (1999) did not find a significant alteration in mRNA levels of genes coding for factors involved in mtDNA replication when cells were depleted of mtDNA. Therefore, the available information suggests that mtDNA levels do not seem to influence transcriptional expression of the known mtDNA replication-related genes.

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Because of their observations showing that cells maintain a certain mass of mitochondrial genomes, Tang et al. (2000) suggested that the regulation of mtDNA copy number could be provided by the control of the organellar nucleoside pools. Although cellular nucleotide pools are tightly regulated, defects in nucleotide metabolism are associated with human diseases and mtDNA stability (Nishino et al., 1999; Kaukonen et al., 2000). Control of the available dNTP pools inside mitochondria is known to be important for replication fidelity (Kunkel and Alexander, 1986; Wernette et al., 1988) and depends on factors that regulate nucleotide metabolism such as mitochondrial deoxyribonucleotidases (Rampazzo et al., 2000), which can also offer another level of regulation. Evidence suggesting that mtDNA maintenance and copy-number control depend on factors other than nucleotide pools or housekeeping replication factors comes from the observation of Shoubridge and colleagues (Jenuth et al., 1997) that in some mouse tissues there is a tissue-specific and age-related directional selection for different mtDNA genotypes. This suggests the presence of tissue-specific nuclear genes important for mtDNA maintenance. Moraes et al., (1999) found that ape mtDNA (gorilla or chimpanzee mtDNA) could repopulate human cells devoid of mtDNA (po cells) at a rate similar to wild-type human mtDNAs. However, ape mtDNA was not maintained in human cells harboring wild-type or defective human mtDNA (either with a large deletion or a point mutation). These observations suggested that competition between the two haplotypes prevented the maintenance of ape genomes, underscoring the importance of recognition of the mtDNA primary sequence by cognate replication factors.

IX. Mitochondrial Transcription

A. TRANSCRIPTIONINITIATION There are two major transcription initiation sites in the human mtDNA D-loop region (termed OH and OL) situated within 150 bp of one another (Fig. 1). A promoter element with a 15-bp sequence motif, 5'-CANACC(G) CC(A)AAAGAN, surrounds the transcription initiation sites and is necessary for transcription (Chang and Clayton, 1984). Heavy-strand transcription starts at nucleotide position 561, located within the H-strand promoter (HSP) and flanked by the tRNAPhe gene, whereas L-strand transcription starts at nucleotide position 407, within the LSP (Fig. 1). Despite the close proximity of the HSP and LSP, the initial in vitro transcription studies demonstrated that these elements are functionally independent (Chang and Clayton, 1984; Hixson and Clayton, 1985; Topper

MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

and Clayton, 1989). This functional autonomy was confirmed in patients with progressive external ophthalmoplegia, with mutated muscle mtDNA harboring large-scale deletions including the HSE In affected muscle cells, LSP was fully active (Moraes et al., 1991). A second initiation site for H-strand transcription has been described around nucleotide position 638, adjacent to the gene for 12S rRNA. Its promoter region only shows limited similarity with the 15-bp consensus sequence and is used less frequently for transcription of the H-strand (Montoya et al., 1983). Fractionation of human mitochondrial transcription extracts showed the requirement for at least two factors for transcriptional activity: (1) a relatively nonselective core RNA polymerase and (2) a dissociable transcription factor that confers promoter selectivity to the polymerase (Fisher et al., 1987). A human cDNA specifying the mitochondrial RNA polymerase was identified by screening of an expressed sequence tags (EST) database with the yeast sequence (Tiranti et al., 1997). It was found that the C-terminal half of the predicted polypepfide shares significant amino acid sequence identity with the single subunit RNA polymerases of T3, T7, and SP6 bacteriophages. A mitochondrial transcription factor was identified by Clayton and colleagues (Fisher and Clayton, 1985). This factor, currently known as mtTFA (or Tfam) is a 25-kDa mitochondrial protein that contains two high mobility group (HMG) domains separated by a 27-amino acid residue linker and followed by a 25-amino acid residue basic C-terminal tail. HMG domains are involved in DNA binding, and are found in a diverse family of proteins whose members have been implicated in processes such as transcription enhancement and chromatin packaging. Mutation analysis of the human mtTFA has demonstrated that its C-terminal tail is important for specific DNA recognition and is essential for efficient transcription (Dairaghi et al., 1995a). The mechanism of transcription stimulation by mtTFA seems to be related to its ability to bend DNA upon binding, thereby facilitating DNA strands unwinding (Fisher et al., 1992). Scanning transmission electron microscopy revealed that the Xenopus homologue also causes sharp bending of the DNA duplex at the promoter activation site (Antoshechkin et al., 1997). These mtTFA-induced conformational changes of mtDNA may be required to allow the core RNA polymerase access to the template for initiation of the transcription process. As described above, both major transcription promoters in human mitochondria can function bidirectionaUy, in vitro as well as in vivo (Chang et al., 1986). The asymmetric binding of mtTFA relative to the transcription start site may ensure that transcription proceeds primarily in a unidirectional fashion (Fig. 1). The existing 10-bp spacing between the mtTFA binding site and the start site of transcription seem to be necessary for efficient transcription (Dairaghi et al., 1995b).

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An additional protein, which is necessary for mitochondrial RNA polymerase activity, has been identified in yeast and XenqOus laevis (Antoshechkin and Bogenhagen, 1995; Lisowsky and Michaelis, 1988). This factor, designated mtTFB, exhibits sequence homology to the dissociable subunit of bacterial RNA polymerases which is responsible for promoter recognition. More recently, McCulloch and colleagues (2002) and Falkemberg and colleagues (2002) identified this second mitochondrial transcription factor in h u m a n cells (mtTFB or TFBM). The latter study identified two forms of this factor (TFB1M and TFB2M) with TFB2M having the strongest activity. These proteins have homology to rRNA dimethyltransferases and RNA adenine methyltransferases suggesting that they are part of a family of nucleic acid binding proteins. Although they seem to interact with the mitochondrial RNA polymerase, their mode of action remains to be determined.

B. TRANSCRIPTIONELONGATIONAND TERMINATION The mtDNA L-strand is transcribed as a single polycistronic precursor RNA, encompassing most of the genetic information encoded on the strand (Murphy et al., 1975; Montoya et al., 1981). The HSP seems to direct transcription of the entire H-strand in a similar fashion. In all studied cells, the rRNAs are synthesized at a much higher rate than the individual mRNAs encoded on the H-strand (Gelfand and Attardi, 1981). This difference has been explained by two possible mechanisms: (1) the existence of two distinct initiation sites for H-strand transcription. According to this model, transcription starts relatively frequent at the first HSP and then terminates at the downstream end of the 16S rRNA gene. This transcription process would be responsible for synthesis of the vast majority of the two rRNA species. In contrast, transcription starting at a second HSP would be less frequent but would result in polycistronic molecules corresponding to almost the entire H-strand, yielding all the mRNAs and most of the tRNAs encoded on the H-strand (Montoya et al., 1982). (2) the difference in synthesis rate of rRNA/mRNA could also be explained by transcription attenuation at the junction of the 16S rRNA and tRNA Leu(t~UR) genes by a factor named mTERF (Kruse et al., 1989) (or mtTERM; Hess et al., 1991). In addition to an attenuation function for H-strand transcription, mTERF may also stop L-strand transcription at a site where no L-strand-encoded genes are found downstream (Hess et al., 1991). Because mTERF also mediates termination of transcription by heterologous RNA polymerases, it probably stops elongation of transcription by constituting a physical barrier, rather than by specific interactions with the RNA polymerase, (Shang and Clayton, 1994). These two mechanisms are not mutually exclusive, and may work in a coordinated manner.

MITOCHONDRIAL DNA STRUCTURE AND FUNCTION

A polypeptide of around 34 kDa has been associated with mTERF function. The cDNA coding for the human polypeptide was cloned and sequenced. The polypeptide contains two widely separated basic regions and three leucine zipper motifs that were necessary for its DNA-binding capacity (Fernandez-Silva et al., 1997). However, the recombinant protein was unable to promote transcription termination in an in vitro system, suggesting that an additional component may be required for the termination activity.

C. POSTTRANSCRIPTIONAL MODIFICATIONS

No intron sequences are present in vertebrate mtDNA and intergenetic sequences are minimal. The processing of the long polycistronic H- and L-strand messengers is thought to require only a few enzymes. Genes for tRNAs flank the two rRNA genes and nearly every protein coding gene (Fig. 1). This unique genetic organization has suggested that the secondary structure of the tRNA sequences provide "cleavable tags" (Ojala et al., 1981). Precise endonucleolyfic excision of the tRNAs from the nascent transcripts will concomitantly yield correctly processed rRNAs, and in most cases, correctly processed mRNAs (Ojala et al., 1981; Montoya et al., 1982). In cases in which the mRNA termini cannot be accounted for by tRNA excision, the processing enzyme possibly recognizes a secondary structure that shares features with the cloverleaf structures of tRNAs. Mitochondrial mRNAs are polyadenylated by a mitochondrial poly(A) polymerase during or immediately after cleavage (Rose et al., 1975). Maturation of mitochondrial tRNAs involves three enzymatic activities: (1) cleavage at the 5' end. This activity is performed by a mitochondrial RNase P (mtRNase P). In contrast to vertebrates' mtRNase P, yeast mtRNase P has been characterized in detail. The enzyme of S. cerevisiae is composed of a nuclear-encoded protein and a mtDNA-encoded RNA species (Dang and Martin, 1993). The RNA moiety of the ribonucleoprotein complex is AU-rich and forms the catalytic core of the enzyme. (2) Cleavage at the 3' end. The endonuclease responsible for 3' end cleavage of tRNAs has not been characterized. (3) Maturation of the excised tRNAs. This process is completed by addition of the sequence CCA to their 3' end catalyzed by ATP(CTP) :tRNA nucleotidyltransferase (Rossmanith et al., 1995).

X. Translation of Mitochondrial Transcripts

There are close to 100 ribosomes per mitochondrion (Cantatore et al., 1987). Mammalian mitochondrial ribosomes have an unusually low RNA

MORAES et al.

content, and consequently, a low sedimentation coefficient (approximately 55S (Hamilton and O'Brien, 1974)). The 39S and 28S ribosomal subunits contain the 16S and 12S rRNA species, respectively, which are encoded by the mtDNA (Attardi and Ojala, 1971; Brega and Baglioni, 1971). Twodimensional gel electrophoresis has allowed the identification of 85 protein spots from bovine and 86 from rodent mitochondrial ribosomes (Matthews et al., 1982; Cahill et al., 1995). It is possible that some of these mitochondrial ribosomal proteins have adopted structural and functional roles of rRNA sequences. Although mammalian mitochondrial ribosomes differ in many aspects from both eukaryote (cytosolic) and prokaryote ribosomes, they retain some properties of the putative prokaryote ancestral, such as sensitivity to chloramphenicol and insensitivity to cyclohexemide (Lamb et al., 1968). The mitochondrial rRNA and tRNA species are relatively small when compared to other systems. Mammalian mitochondrial mRNAs have no 5' untranslated region (5' UTR) and are devoid of a cap structure (Grohmann et al., 1978). Because of this lack of 5' UTR, coding sequences start at or very near the 5' end with the codon for the initiating N-formylmethionine (Montoya et al., 1981). Approximately 400 nucleotides are required for efficient binding of mRNAs to the small ribosomal subunit, even though a smaller region actually interacts with the ribosome (Denslow et al., 1989; Liao and Spremulli, 1989; Liao and Spremulli, 1990). After binding of the small ribosomal subunit to the messenger, the subunit appears to move to the 5' end of the mRNA mediated by yet unknown auxiliary initiation factors (Denslow et al., 1989). The only initiation factor identified in mammalian mitochondria to date is mtIF-2 (Liao and Spremulli, 1991). The cDNA for the human mtIF-2 has been cloned and sequenced (Ma and Spremulli, 1995). This monomeric protein belongs to the family of GTPases and promotes fMet-tRNA binding to the small ribosomal subunit in the presence of GTP and a template. Detailed in vitro characterization of bovine mtIF-2 suggested that mtIF-2 may bind to the small ribosomal subunit prior to its interaction with GTP. GTP would enhances the affinity between mtIF-2 and the small subunit and allow fMettRNA to join the complex (Ma and Spremulli, 1996). Hydrolysis of GTP seems to facilitate the release of mtIF-2 and the association of the large (39S) ribosomal subunit to form the 55S initiation complex. Nonhydrolysable analogues of GTP can still promote formation of the initiation complex, indicating that GTP hydrolysis is not required for subunitjoining (Liao and Spremulli, 1991). The mitochondrial elongation factors, mtEF-Tu, mtEF-Ts, and mtEF-G, have been purified from bovine liver (Schwartzbach and Spremulli, 1989; Chung and Spremulli, 1990). The human cDNAs for all three factors have

MITOCHONDRIALDNA STRUCTUREAND FUNCTION b e e n c l o n e d a n d s e q u e n c e d (Ma a n d Spremulli, 1995; Woriax et al., 1995; X i n et al., 1995). T h e in vitro characterization o f the purified factors a n d the c D N A s e q u e n c e i n f o r m a t i o n have revealed similarities with the corr e s p o n d i n g prokaryotic factors, suggesting that e l o n g a t i o n o f the n a s c e n t m i t o c h o n d r i a l p o l y p e p t i d e in m i t o c h o n d r i a p r o c e e d s in a similar fashion as in bacterial systems (Nierhaus, 1996).

Xl. ConcludingRemarks

Because o f its p r o b a b l e p r o k a r y o t e origin, in m a n y aspects, the mitoc h o n d r i o n behaves as an i n d e p e n d e n t entity living inside an eukaryotic cell. All basic processes associated with life (DNA m a i n t e n a n c e , transcription, a n d translation) o c c u r inside the organelle. However, the vast majority o f the factors involved in p r o m o t i n g a n d c o n t r o l l i n g these processes are borr o w e d f r o m the cytoplasm, w h e r e n u c l e a r - c o d e d proteins are synthesized. O u r u n d e r s t a n d i n g o f the intricate relationships between m i t o c h o n d r i a l a n d n u c l e a r g e n o m e s is still limited, d u e mainly to the fact that genetic m a n i p u l a t i o n a n d in vitro systems are difficult to develop for organelles. Nevertheless, a picture is e m e r g i n g that shows the m i t o c h o n d r i a l genetic system has m a n y features in c o m m o n with the putative p r o k a r y o t e ancestral, b u t yet has d e v e l o p e d a n u m b e r o f u n i q u e m e c h a n i s m s as it evolved as an e n d o s y m b i o n t taking advantage o f w h a t a c o m p l e x eukaryote n u c l e a r g e n o m e has to offer.

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