- Email: [email protected]
Organelle DNA
128
TIBS - April 1984
Organelle DNA Piet Borst, Leslie A. Griveil and Gert S. P. Groot Mitochondria and chloroplasts contain their own genetic systems which make an essential contribution to the biosynthesis of these organelles. Study of organelle DNA has considerably widened our perspectives on the basic mechanisms of gene expression. Prehistoryt,2 Why study organelle biogenesis? First "and foremost to find out why mitochondria and chloroplasts contain their own genetic system, whereas organelles like peroxisomes can be made without. Second, to unravel the complex interplay between organelle and nucleus in the joint venture that results in the construction of organelles; and, finally, in the hope that organelle genetic systems may provide new insights in basic mechanisms of gene expression, such simple systems being able to operate with 'no-frills' machinery. Little had been realized of these expectations way back in 1976 when TIBS began. We had a rudimentary knowledge of the size, structure and complexity of organelle DNA; the translational machinery had been roughly characterized; several of the organelle genes had been identified by genetics and their gene products by analysis of the proteins made in organelles. The first piece of mtDNA, a 68-bp segment of yeast mtDNA amplified in a petite mutant, had just been sequenced via laborious primed RNA copying methods. However, introns had not been discovered; it was largely unknown how RNA transcripts were generated; it was unclear how organelle proteins encoded in the nucleus found their way into the organelle. The genetic code was still universal. What has happened since 1976? • The contribution of organdie DNA to organdie biosynthesis a-9 In the past eight years, new DNA sequencing techniques have rapidly advanced our knowledge of organelle genes. A landmark was the publication of the complete sequence of the 16 569 base pairs of human mtDNA, published Piet Borst is at the Department of Molecular Biology, Amoni van Leeuwenhoekhuis, Netherlands Cancer" Institute, Plesmanlaan 121. 1066 C X Amsterdam, The Netherlands. Leslie A. Grivell is at the Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam. The Netherlands. Gert S. P. Groot is at the Laboratory o f Biochemistry, Free University, de B oelelaan 1083, 1081 H V Amsterdam, The Nethertdnds.
in 1981t°. Complete or extensive sequences are now also available for mouse, rat, Drosophila, yeast, Aspergillus, Neurospora and Trypanosoma brucei mtDNAs and chloroplast DNAs from algae and higher plants. Three points should be noted from the representative gene complements presented in Tables I and II. (1) The early observation that many enzyme complexes of organelles contain gene products of both organelle and nuclear genes remains a striking feature. Chloroplast ribulose-bisphosphate carboxylase contains one imported subunit and one subunit specified by chloroplast DNA. The ribosomal RNAs (rRNAs) of organelles are specified by the organelle genome, but most ribosomal proteins are imported. Similar dual origins have been found for the subunits of the mitochondrial and chloroplast electron transport and ATPase complexes. Interestingly, the division of labour between organelle and nuclear genome varies in different organisms and organelles, as illustrated by the ATPase complex. Although the composition of the complex has been strongly conserved during evolution, the contri-
bution of organelle DNA to its synthesis has not. In animal mitochondria only two subunits of the membrane part are encoded in mtDNA, the remainder including some very hydrophobic subunits - must be imported. In yeast, three membrane subunits are made in mitochondria; in maize mitochondria at least one of the subunits of the nonmembrane FI part is also made in mitochondria; and in chloroplasts the majority of the subunits are specified by organelle genes. (2) Many of the open reading frames found in mtDNAs remain unassigned to known proteins (URFs). All of these URFs are highly conserved between mouse and man and one of the mammalian URFs has strong homology with a URF from the, fungus Aspergillus. Some of the URF gene products may be involved in RNA processing, as has been proven for tl~e 'maturases' encoded in the introns of the apocytochrome b (COB) gene of yeast. (3) Whereas all chloroplast DNAs are between 120 and 200 kb and in most plants are organized in a very similar way, mtDNAs are more diverse in size, ranging from the 15 kb circles found in animals to the 600-750 kb of many higher plants. Much of this diversity is due to differences in packing efficiency. Animal mtDNAs 'look like a 1981 University Department, with everything reduced to minimal size without anyone actually being fired'H. In yeast, the equivalent set of genes is spread luxuriously over 78 kb, mainly due to the
Table I. An inventory of mitochondrial genes Mitochondrial component
Cytochrome c oxidase Subunit I Subunit II Subunit III Ubiquinol cytochrome c reductase Apocytochrome b ATPase complex Subunit 6 Subunit 9 Subunit 8 F1 subunit a Large ribosomal subunit
rRNA Small ribosomal subunit rRNA Ribosome-assoc. protein R N A processing enzymes Intron 2 COB maturase Intron 4 C O B maturase tRNAs URFs Adapted from Ref. 4.
~) 1984,ElsevierSciencePublishersB.V.. Amsterdam 0376- 5067/84¢$02.00
Mitochondrial gene product in Yeast
Man
Maize
+ + +
+ + +
+ + +
+
+
+
+ + + -
+ + -
+ +? ? +
+
+
+
+ +
+ -?
+ ?
+ + 25 >--8
22 8
? ? ? ?
TIBS - April 1984 presence of large expanses of AT-rich sequences between genes and large introns within them. Unusual features of the organelle genetic
system4,5,8,9 No major surprises have emerged from the analysis of the chloroplast genetic system. This looks like a shrivelled bacterial system and many components will even work well in reconstituted bacterial systems. In contrast, recent work on mitochondria has yielded a plethora of unusual results. These have contributed novel insights into genetic control mechanisms 'and have substantially lowered our assessment of the minimal requirements for functional components of a genetic system. Examples are: (1) Genetic code. In many organisms the mitochondrial genetic code differs from the 'universal' code and different organisms use different variants. The most common deviation is the use of U G A for Trp rather than stop, but even this is not a common feature of all mitochondria, because higher plants probably adhere to the 'universal' code in this respect. Most investigators now agree that these divergent codes reflect evolutionary accidents, which may be relatively harmless in a genetic system that encodes so few proteins. (2) tRNAs. The wobble rules deduced by Crick predict that an organism requires at least 32 different tRNAs to faithfully read all codons. Animal mitochondria have managed to devise tRNAs that can read four synonymous codons, however, and they get away with only 22 tRNAs. Some of these conform to the general structure of tRNAs in other genetic systems, but others are remarkably bizarre, like one of the mammalian tRNAs for serine, which lacks one arm. (3) rRNAs. In chloroplasts and higher plant mitochondria the rRNAs still have a clear homology with bacterial rRNAs and also include a typical 5S RNA. A 5S R N A is missing from mitochondrial ribosomes of other branches of the evolutionary tree and, moreover, the size of the two large rRNAs has often shrunk. The most extreme case thus far comes from Trypanosoma brucei, where the size is down to 1150 and 600 nucleotides and the small rRNA has no significant sequence homology with any other rRNA. Another interesting feature of mitochondrial ribosomes in animals and yeast is the existence of point mutations in the gene for the large r R N A that confer highlevel resistance to inhibitors of the peptidyl transferase, like chloramphenicol
129 Table II.
Genes on
chloroplast DNA
Stuctural RNA genes 35-40 tRNAs 1-5 sets of rRNA Genes for ribosomal proteins
Approximately20 (includinggenes homologous to the genes of E. coli ribosomalproteins L2, $4, $7, S12 and S19) Genes for membraneproteins ATPase complex:3 subunits (out of 5) of the CF~part; at leastone subunit (out of 3) of the CFopart. PhotosystemII: two chlorophyll-conjugated polypeptides,the 32 kD herbicide-binding protein and cytochromeb559apoprotein. PhotosystemI: P700chlorophylla-conjugated apoprotein. Cytochromebr/f: 3 subunits includingthe apoproteinsof cytochrorneb 6 and cytochromef. Genes for stromal polyproteins Large subunit of ribulose-bisphosphate carboxylase. Elongationfactors G~and T. aNot in Euglena.
Adapted from Ref. 4. and erythromycin. The mutations conferring resistance to chloramphenicol fall mainly in a 10-bp segment of the gene that is identical in Escherichia coli, yeast and animal mitochondria. The presence of multiple genes for r R N A probably prevents the emergence of such mutants in bacteria. (4) Introns 12. In contrast to their tightly-packed bretheren in animal mtDNAs, several genes in yeast and other fungal mtDNAs are split by introns. None of these conform to the G T , . . A G consensus and many of them are unusual in containing long open reading frames. Some of these URFs have been shown to encode a protein that, in combination with nuclearcoded splicing enzymes, is required for splicing of the intron specifying it. The other URFs may have a similar function. Synthesis of these proteins, called R N A maturases, and the splicing of the corresponding introns are thus basically self-regulatory processes. As a consequence, levels of R N A maturases in normal cells are vanishingly low and not o n e has yet been isolated in active form. Most things we know of these intriguing proteins have, therefore, been deduced from their D N A sequence. Sequence comparison of mitochondrial introns reveals a striking degree of homology and similarities in potential R N A secondary structure. Each can be assigned to either of two families and it seems reasonable to suppose that each has arisen by duplication and transposition of an ancestral gene. The evolutionary conservation of secondary structure has led to the idea that intron folding promotes splicing by making intron ends meet. This idea has received
support from the finding that sequences identified by mutations as being important for splicing either form part of the predicted secondary structure or are brought together by folding. All in all, the requirements for intronencoded maturases, nuclear-coded splicing enzymes and intron folding show that mitochondrial splicing reactions are remarkably complex events. Why should this be? One idea is that the mechanisms involved are primitive ones and that a 'belt-and-braces' approach is necessary to maintain both accuracy and specificity. It is interesting in this context that vestigial homologies in both sequence and folding can be detected between mitochondrial introns and certain introns encoded by chloroplast or nuclear DNA. More sophisticated or streamlined mechanisms of splicing, perhaps even including that of 'self splicing', may, therefore, derive from mechanisms used by mitochondria. (5) Transcription 13'14. Even without splicing, transcripts in mitochondria seem to lead a complicated life. Mammalian mtDNAs contain only a single major promoter area in each strand. As a result, primary transcripts are of full genomic length and individual gene transcripts are generated by processing at the t R N A sequences that flank virtually every gene. Transcriptional attenuation at a site directly downstream of the r R N A genes is used to achieve an almost 100-fold difference in the level of ribosomal transcripts relative to those of other genes and it is interesting that the choice between attenuation and readthrough seems to be determined by the site used for initiation of transcription. A start at the major promoter yields early termination and synthesis of rRNAs; a start at a closely-adjacent minor promoter yields full-length transcripts. How the effects of these different start points are transmitted to the termination site some 2675 bp downstream is still largely unclear, however. The discovery of multiple initiation sites for R N A synthesis in yeast mitochondria suggests that the scope for transcriptional controls is greater than in animal mitochondria, though as yet little is known of the mechanisms leading to differential gene expression. R N A processing is important here too, since in several cases groups of genes belong to the same large transcriptional unit and mature transcripts are generated by extensive processing. Import of protein into organelles nuclear genes 7'~5'16 Only a small fraction of the total
T I B S - A p r i l 1984
130 number of organelle proteins is encoded instance, apocytoehrome b and the and synthesized within the organelles three larger subunits of cytochrome c themselves. All other proteins are speci- oxidase are encoded in mtDNA in all fied by nuclear genes, synthesized in the organisms analysed. Why should this cytoplasm and imported. As shown be? An evolutionary accident is unlikely, initially for chloroplasts, import is a because the investment involved in post-translational event. Most proteins maintaining a separate genetic system is are synthesized with amino-terminal substantial. If the URFs are disregarded, extensions, which are cleaved proteo- then the mitochondrial genetic system in lytically after transport. With a few animal cells apparently exists for the notable exceptions, uptake is dependent sole purpose of making only six proteins on a membrane potential, which could (Table I). These six proteins show the facilitate protein movement by labilizing maternal inheritance characteristic of the lipid bilayer. How proteins are mtDNA in animal cells and will probably specifically addressed to either mito- show all the other peculiarities associated chondrion or chloroplast is still an un- with cytoplasmic genetics, i.e. homosolved problem. Current models assume zygosity and rapid segregation of the presence of characteristic topogenic mutants 17. Whether any of these sequences within the proteins concerned peculiarities make it advantageous to that can be recognized by membrane- have the genes for these proteins in bound import receptors. As yet, mtDNA rather than in the nucleus characterization of such receptors is in remains an unresolved question. the earliest stages, while nothing at all is known about the topogenic elements Some remaining questions they recognize. Further resolution and Some of the basic questions that reconstitution of in vitro import systems remain have already been touched upon. coupled with site-directed mutagenesis How is the transcription of organelles of import proteins should bring clarifi- controlled? Why are some genes in organelle D N A and others in the cation in the none too distant future. nucleus? How do genes in organelle a n d nucleus work together in making an Why organelle DNA? Tables I and II show why organelle organelle? Why do genes in mtDNA D N A is essential for present-day evolve more rapidly than genes in nuclear eukaryotes. The real question, how- DNA in the same animal cell? Why are ever, is how the responsibility of making some organelle genetic systems, e.g. organelles came to be distributed be- mtDNAs of trypanosomes or higher tween nucleus and organelle DNA and plants, so weird (and how weird exactly)? Progress has been enormous since why the organelle genetic system has survived till today. The resemblance of 1976, but the mopping-up phase is still this system to bacterial systems, espe- not in sight. As more organelle genomes cially in chloroplasts and in the mito- are sequenced and the transcription chondria of higher plants, makes most maps become finished, emphasis will experts believe that organelies originated switch to organelle gene evolution, to in evolution as endosymbionts. The the use of organelle DNA as a tool in ability of nuclear D N A to incorporate genetic and epidemiological studies and any exogenous DNA that wanders in, to the nuclear genes that control could explain the transfer of organelle organelle biogenesis. Further study of genes to the nucleus in the course of the unusual organelle genomes - like evolution. In fact, bits of organelle the mtDNAs from protozoa or higher DNA are even found in nuclear DNA plants - may continue to deepen our today in some organisms, suggesting insight into the minimal requirements that the postulated gene transfer is still and vagaries of a functional genetic possible in present-day eukaryotes. The system and, we hope, demolish additional discovery of introns in genes of archae- cherished dogmas. bacteria has also removed one of the What also remain are questions conlast objections against the endosymbiont ceming the influence of organelle hypothesis for organelle biosynthesis. genomes on the remainder of the cell. If pathways exist for reshuffling of The idea that organelles may code for genes between organelle and nucleus, it proteins that are exported to influence is also understandable why the gene the surrounding cell has been proposed complement present in the organelle repeatedly, but it has never found much may have an element of chance in it, favour and the hard data to support it homologous subunits being encoded in have remained elusive. However, the organelle DNA in one organism and in recent finding of a cytoplasmic genetic the nucleus in another. Nevertheless, factor (probably mtDNA) affecting the the distribution is not random. For expression of nuclear genes of the major
histocompatibility complex is, has refuelled interest in this possibility. A final point concerns the manipulation of organdies in intact cells. When all the sequences required for protein import are known (and all those that are prohibited), it will be possible to redirect proteins normally located outside organelles into mitochondria or chloroplasts and in this way redesign metabolism. One should also be able to transfer all mitochondrial genes to the nucleus (switching codes). This will test whether some of the veteran mitochondrial protein-coding genes specify amino acid sequences that are essential for function but incompatible with import. If these do not exist, it should eventually become possible to make mitochondria without mitochondrial DNA. Clearly there is enough to do for the next 100 issues of T I B S to cover.
References 1 Borst, P. (1977) Trends Biochem. Sei. 2, 31-34 2 Borst, P. (1977) in International Cell Biology,
3 4
5 6
1976--1977(Brinkley,B. R. and Porter,,,K.R., eds), pp. 237-244,The RockefellerUniversity Press, New York, Borst, P. and Grivell, L. A. (1978) Cell 15, 705--723 Borst, P., Tabak, H. F. and Grivell, L. A. (1983) in Eukaryotic Genes: Their Structure, Activity and Regulation (Mactean,N., Gregory, S. P. and Flavell, R. A., eds), pp. 71-84, Butterworths Grivell,L. A. (1983)Sci. Am. 248, 78--89 Dujon, B. (1981)in Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance
(Strathem, J. N., Jones, E. W. and Broach, J. R., eds), pp. 505--635,Cold SpringHarbor Laboratory, Cold SpringHarbor 7 Schweyen,R. J., Wolf, K. and Kaudewitz,F. (eds) (1983)Mitochondria 1983: Nucleo-Mitochondrial Interactions, De Gruyter 8 Whitfeld, P. R. and Bottomley, W. (1983) Ann. Rev. Plant Physiol. 34, 279--310 9 Bohnert, H. J., Crouze, E. J. and Schmitt, J. M. (1982) in Encyclopedia of Plant Physiology: Nucleic Acids and Proteins in Plants (Boulter, D. and Parthier, B., eds), Vol. 14B, pp. 475-530, Springer 10 Anderson, S., Bankier, A. T., Barrell, B. G., De Bruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Staden, R. and Young, I. G. (1981) Nature 290, 457-465 11 Borst, P. and Grivell, L. A. (1981) Nature 290, 443-444 12 Grivell, L. A., Bonen, L. and Borst, P. (1983) in Genes: Structure and Expression (Kroon, A. M., ed.), pp. 279-306, Wiley 13 Tabak, H. F., Grivell, L. A. and Borst, P. (1983) CRC Crit. Rev. Biochem. 14, 297-317 14 Attardi, 13. (1981) Trends Biochem. Sci. 6, 86-89; 100-103 15 Neupert, W. and Schatz, G. (1981) Trends Biochem. Sci. 6, 1--4 16 Schatz, G. and Butow, R. A. (1983) Cell 32, 316-318 17 Birky, C. W. (1983) Science 222,468--475 18 Goodfellow, P. (1983) Nature 306, 539-5z10
TIBS - April 1984
Organelle DNA Piet Borst, Leslie A. Griveil and Gert S. P. Groot Mitochondria and chloroplasts contain their own genetic systems which make an essential contribution to the biosynthesis of these organelles. Study of organelle DNA has considerably widened our perspectives on the basic mechanisms of gene expression. Prehistoryt,2 Why study organelle biogenesis? First "and foremost to find out why mitochondria and chloroplasts contain their own genetic system, whereas organelles like peroxisomes can be made without. Second, to unravel the complex interplay between organelle and nucleus in the joint venture that results in the construction of organelles; and, finally, in the hope that organelle genetic systems may provide new insights in basic mechanisms of gene expression, such simple systems being able to operate with 'no-frills' machinery. Little had been realized of these expectations way back in 1976 when TIBS began. We had a rudimentary knowledge of the size, structure and complexity of organelle DNA; the translational machinery had been roughly characterized; several of the organelle genes had been identified by genetics and their gene products by analysis of the proteins made in organelles. The first piece of mtDNA, a 68-bp segment of yeast mtDNA amplified in a petite mutant, had just been sequenced via laborious primed RNA copying methods. However, introns had not been discovered; it was largely unknown how RNA transcripts were generated; it was unclear how organelle proteins encoded in the nucleus found their way into the organelle. The genetic code was still universal. What has happened since 1976? • The contribution of organdie DNA to organdie biosynthesis a-9 In the past eight years, new DNA sequencing techniques have rapidly advanced our knowledge of organelle genes. A landmark was the publication of the complete sequence of the 16 569 base pairs of human mtDNA, published Piet Borst is at the Department of Molecular Biology, Amoni van Leeuwenhoekhuis, Netherlands Cancer" Institute, Plesmanlaan 121. 1066 C X Amsterdam, The Netherlands. Leslie A. Grivell is at the Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam. The Netherlands. Gert S. P. Groot is at the Laboratory o f Biochemistry, Free University, de B oelelaan 1083, 1081 H V Amsterdam, The Nethertdnds.
in 1981t°. Complete or extensive sequences are now also available for mouse, rat, Drosophila, yeast, Aspergillus, Neurospora and Trypanosoma brucei mtDNAs and chloroplast DNAs from algae and higher plants. Three points should be noted from the representative gene complements presented in Tables I and II. (1) The early observation that many enzyme complexes of organelles contain gene products of both organelle and nuclear genes remains a striking feature. Chloroplast ribulose-bisphosphate carboxylase contains one imported subunit and one subunit specified by chloroplast DNA. The ribosomal RNAs (rRNAs) of organelles are specified by the organelle genome, but most ribosomal proteins are imported. Similar dual origins have been found for the subunits of the mitochondrial and chloroplast electron transport and ATPase complexes. Interestingly, the division of labour between organelle and nuclear genome varies in different organisms and organelles, as illustrated by the ATPase complex. Although the composition of the complex has been strongly conserved during evolution, the contri-
bution of organelle DNA to its synthesis has not. In animal mitochondria only two subunits of the membrane part are encoded in mtDNA, the remainder including some very hydrophobic subunits - must be imported. In yeast, three membrane subunits are made in mitochondria; in maize mitochondria at least one of the subunits of the nonmembrane FI part is also made in mitochondria; and in chloroplasts the majority of the subunits are specified by organelle genes. (2) Many of the open reading frames found in mtDNAs remain unassigned to known proteins (URFs). All of these URFs are highly conserved between mouse and man and one of the mammalian URFs has strong homology with a URF from the, fungus Aspergillus. Some of the URF gene products may be involved in RNA processing, as has been proven for tl~e 'maturases' encoded in the introns of the apocytochrome b (COB) gene of yeast. (3) Whereas all chloroplast DNAs are between 120 and 200 kb and in most plants are organized in a very similar way, mtDNAs are more diverse in size, ranging from the 15 kb circles found in animals to the 600-750 kb of many higher plants. Much of this diversity is due to differences in packing efficiency. Animal mtDNAs 'look like a 1981 University Department, with everything reduced to minimal size without anyone actually being fired'H. In yeast, the equivalent set of genes is spread luxuriously over 78 kb, mainly due to the
Table I. An inventory of mitochondrial genes Mitochondrial component
Cytochrome c oxidase Subunit I Subunit II Subunit III Ubiquinol cytochrome c reductase Apocytochrome b ATPase complex Subunit 6 Subunit 9 Subunit 8 F1 subunit a Large ribosomal subunit
rRNA Small ribosomal subunit rRNA Ribosome-assoc. protein R N A processing enzymes Intron 2 COB maturase Intron 4 C O B maturase tRNAs URFs Adapted from Ref. 4.
~) 1984,ElsevierSciencePublishersB.V.. Amsterdam 0376- 5067/84¢$02.00
Mitochondrial gene product in Yeast
Man
Maize
+ + +
+ + +
+ + +
+
+
+
+ + + -
+ + -
+ +? ? +
+
+
+
+ +
+ -?
+ ?
+ + 25 >--8
22 8
? ? ? ?
TIBS - April 1984 presence of large expanses of AT-rich sequences between genes and large introns within them. Unusual features of the organelle genetic
system4,5,8,9 No major surprises have emerged from the analysis of the chloroplast genetic system. This looks like a shrivelled bacterial system and many components will even work well in reconstituted bacterial systems. In contrast, recent work on mitochondria has yielded a plethora of unusual results. These have contributed novel insights into genetic control mechanisms 'and have substantially lowered our assessment of the minimal requirements for functional components of a genetic system. Examples are: (1) Genetic code. In many organisms the mitochondrial genetic code differs from the 'universal' code and different organisms use different variants. The most common deviation is the use of U G A for Trp rather than stop, but even this is not a common feature of all mitochondria, because higher plants probably adhere to the 'universal' code in this respect. Most investigators now agree that these divergent codes reflect evolutionary accidents, which may be relatively harmless in a genetic system that encodes so few proteins. (2) tRNAs. The wobble rules deduced by Crick predict that an organism requires at least 32 different tRNAs to faithfully read all codons. Animal mitochondria have managed to devise tRNAs that can read four synonymous codons, however, and they get away with only 22 tRNAs. Some of these conform to the general structure of tRNAs in other genetic systems, but others are remarkably bizarre, like one of the mammalian tRNAs for serine, which lacks one arm. (3) rRNAs. In chloroplasts and higher plant mitochondria the rRNAs still have a clear homology with bacterial rRNAs and also include a typical 5S RNA. A 5S R N A is missing from mitochondrial ribosomes of other branches of the evolutionary tree and, moreover, the size of the two large rRNAs has often shrunk. The most extreme case thus far comes from Trypanosoma brucei, where the size is down to 1150 and 600 nucleotides and the small rRNA has no significant sequence homology with any other rRNA. Another interesting feature of mitochondrial ribosomes in animals and yeast is the existence of point mutations in the gene for the large r R N A that confer highlevel resistance to inhibitors of the peptidyl transferase, like chloramphenicol
129 Table II.
Genes on
chloroplast DNA
Stuctural RNA genes 35-40 tRNAs 1-5 sets of rRNA Genes for ribosomal proteins
Approximately20 (includinggenes homologous to the genes of E. coli ribosomalproteins L2, $4, $7, S12 and S19) Genes for membraneproteins ATPase complex:3 subunits (out of 5) of the CF~part; at leastone subunit (out of 3) of the CFopart. PhotosystemII: two chlorophyll-conjugated polypeptides,the 32 kD herbicide-binding protein and cytochromeb559apoprotein. PhotosystemI: P700chlorophylla-conjugated apoprotein. Cytochromebr/f: 3 subunits includingthe apoproteinsof cytochrorneb 6 and cytochromef. Genes for stromal polyproteins Large subunit of ribulose-bisphosphate carboxylase. Elongationfactors G~and T. aNot in Euglena.
Adapted from Ref. 4. and erythromycin. The mutations conferring resistance to chloramphenicol fall mainly in a 10-bp segment of the gene that is identical in Escherichia coli, yeast and animal mitochondria. The presence of multiple genes for r R N A probably prevents the emergence of such mutants in bacteria. (4) Introns 12. In contrast to their tightly-packed bretheren in animal mtDNAs, several genes in yeast and other fungal mtDNAs are split by introns. None of these conform to the G T , . . A G consensus and many of them are unusual in containing long open reading frames. Some of these URFs have been shown to encode a protein that, in combination with nuclearcoded splicing enzymes, is required for splicing of the intron specifying it. The other URFs may have a similar function. Synthesis of these proteins, called R N A maturases, and the splicing of the corresponding introns are thus basically self-regulatory processes. As a consequence, levels of R N A maturases in normal cells are vanishingly low and not o n e has yet been isolated in active form. Most things we know of these intriguing proteins have, therefore, been deduced from their D N A sequence. Sequence comparison of mitochondrial introns reveals a striking degree of homology and similarities in potential R N A secondary structure. Each can be assigned to either of two families and it seems reasonable to suppose that each has arisen by duplication and transposition of an ancestral gene. The evolutionary conservation of secondary structure has led to the idea that intron folding promotes splicing by making intron ends meet. This idea has received
support from the finding that sequences identified by mutations as being important for splicing either form part of the predicted secondary structure or are brought together by folding. All in all, the requirements for intronencoded maturases, nuclear-coded splicing enzymes and intron folding show that mitochondrial splicing reactions are remarkably complex events. Why should this be? One idea is that the mechanisms involved are primitive ones and that a 'belt-and-braces' approach is necessary to maintain both accuracy and specificity. It is interesting in this context that vestigial homologies in both sequence and folding can be detected between mitochondrial introns and certain introns encoded by chloroplast or nuclear DNA. More sophisticated or streamlined mechanisms of splicing, perhaps even including that of 'self splicing', may, therefore, derive from mechanisms used by mitochondria. (5) Transcription 13'14. Even without splicing, transcripts in mitochondria seem to lead a complicated life. Mammalian mtDNAs contain only a single major promoter area in each strand. As a result, primary transcripts are of full genomic length and individual gene transcripts are generated by processing at the t R N A sequences that flank virtually every gene. Transcriptional attenuation at a site directly downstream of the r R N A genes is used to achieve an almost 100-fold difference in the level of ribosomal transcripts relative to those of other genes and it is interesting that the choice between attenuation and readthrough seems to be determined by the site used for initiation of transcription. A start at the major promoter yields early termination and synthesis of rRNAs; a start at a closely-adjacent minor promoter yields full-length transcripts. How the effects of these different start points are transmitted to the termination site some 2675 bp downstream is still largely unclear, however. The discovery of multiple initiation sites for R N A synthesis in yeast mitochondria suggests that the scope for transcriptional controls is greater than in animal mitochondria, though as yet little is known of the mechanisms leading to differential gene expression. R N A processing is important here too, since in several cases groups of genes belong to the same large transcriptional unit and mature transcripts are generated by extensive processing. Import of protein into organelles nuclear genes 7'~5'16 Only a small fraction of the total
T I B S - A p r i l 1984
130 number of organelle proteins is encoded instance, apocytoehrome b and the and synthesized within the organelles three larger subunits of cytochrome c themselves. All other proteins are speci- oxidase are encoded in mtDNA in all fied by nuclear genes, synthesized in the organisms analysed. Why should this cytoplasm and imported. As shown be? An evolutionary accident is unlikely, initially for chloroplasts, import is a because the investment involved in post-translational event. Most proteins maintaining a separate genetic system is are synthesized with amino-terminal substantial. If the URFs are disregarded, extensions, which are cleaved proteo- then the mitochondrial genetic system in lytically after transport. With a few animal cells apparently exists for the notable exceptions, uptake is dependent sole purpose of making only six proteins on a membrane potential, which could (Table I). These six proteins show the facilitate protein movement by labilizing maternal inheritance characteristic of the lipid bilayer. How proteins are mtDNA in animal cells and will probably specifically addressed to either mito- show all the other peculiarities associated chondrion or chloroplast is still an un- with cytoplasmic genetics, i.e. homosolved problem. Current models assume zygosity and rapid segregation of the presence of characteristic topogenic mutants 17. Whether any of these sequences within the proteins concerned peculiarities make it advantageous to that can be recognized by membrane- have the genes for these proteins in bound import receptors. As yet, mtDNA rather than in the nucleus characterization of such receptors is in remains an unresolved question. the earliest stages, while nothing at all is known about the topogenic elements Some remaining questions they recognize. Further resolution and Some of the basic questions that reconstitution of in vitro import systems remain have already been touched upon. coupled with site-directed mutagenesis How is the transcription of organelles of import proteins should bring clarifi- controlled? Why are some genes in organelle D N A and others in the cation in the none too distant future. nucleus? How do genes in organelle a n d nucleus work together in making an Why organelle DNA? Tables I and II show why organelle organelle? Why do genes in mtDNA D N A is essential for present-day evolve more rapidly than genes in nuclear eukaryotes. The real question, how- DNA in the same animal cell? Why are ever, is how the responsibility of making some organelle genetic systems, e.g. organelles came to be distributed be- mtDNAs of trypanosomes or higher tween nucleus and organelle DNA and plants, so weird (and how weird exactly)? Progress has been enormous since why the organelle genetic system has survived till today. The resemblance of 1976, but the mopping-up phase is still this system to bacterial systems, espe- not in sight. As more organelle genomes cially in chloroplasts and in the mito- are sequenced and the transcription chondria of higher plants, makes most maps become finished, emphasis will experts believe that organelies originated switch to organelle gene evolution, to in evolution as endosymbionts. The the use of organelle DNA as a tool in ability of nuclear D N A to incorporate genetic and epidemiological studies and any exogenous DNA that wanders in, to the nuclear genes that control could explain the transfer of organelle organelle biogenesis. Further study of genes to the nucleus in the course of the unusual organelle genomes - like evolution. In fact, bits of organelle the mtDNAs from protozoa or higher DNA are even found in nuclear DNA plants - may continue to deepen our today in some organisms, suggesting insight into the minimal requirements that the postulated gene transfer is still and vagaries of a functional genetic possible in present-day eukaryotes. The system and, we hope, demolish additional discovery of introns in genes of archae- cherished dogmas. bacteria has also removed one of the What also remain are questions conlast objections against the endosymbiont ceming the influence of organelle hypothesis for organelle biosynthesis. genomes on the remainder of the cell. If pathways exist for reshuffling of The idea that organelles may code for genes between organelle and nucleus, it proteins that are exported to influence is also understandable why the gene the surrounding cell has been proposed complement present in the organelle repeatedly, but it has never found much may have an element of chance in it, favour and the hard data to support it homologous subunits being encoded in have remained elusive. However, the organelle DNA in one organism and in recent finding of a cytoplasmic genetic the nucleus in another. Nevertheless, factor (probably mtDNA) affecting the the distribution is not random. For expression of nuclear genes of the major
histocompatibility complex is, has refuelled interest in this possibility. A final point concerns the manipulation of organdies in intact cells. When all the sequences required for protein import are known (and all those that are prohibited), it will be possible to redirect proteins normally located outside organelles into mitochondria or chloroplasts and in this way redesign metabolism. One should also be able to transfer all mitochondrial genes to the nucleus (switching codes). This will test whether some of the veteran mitochondrial protein-coding genes specify amino acid sequences that are essential for function but incompatible with import. If these do not exist, it should eventually become possible to make mitochondria without mitochondrial DNA. Clearly there is enough to do for the next 100 issues of T I B S to cover.
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