The chloroplast genome was not the first organelle genome to be sequenced completely. The

48 The chloroplast genome was not the first organelle genome to be sequenced completely. The

relatively small size of the human mitochondrial genome made it a particularly attractive target for molecular geneticists equipped with newly devised DNA-sequencing techniques, and in 1981 the complete sequence of its 16,569 nucleotides was published. By comparing this sequence with relatively small size of the human mitochondrial genome made it a particularly attractive target for molecular geneticists equipped with newly devised DNA-sequencing techniques, and in 1981 the complete sequence of its 16,569 nucleotides was published. By comparing this sequence with

Compared to nuclear, chloroplast, and bacterial genomes, the human mitochondrial genome has several surprising features. (1) Unlike other genomes, nearly every nucleotide appears to be part of a coding sequence, either for a protein or for one of the rRNAs or tRNAs. Since these coding sequences run directly into each other, there is very little room left for regulatory DNA sequences. (2) Whereas 30 or more tRNAs specify amino acids in the cytosol and in chloroplasts, only 22 tRNAs are required for mitochondrial protein synthesis. The normal codon-anticodon pairing rules are relaxed in mitochondria, so that many tRNA molecules recognize any one of the four nucleotides in the third (wobble) position. Such "2 out of 3" pairing allows one tRNA to pair with any one of four codons and permits protein synthesis with fewer tRNA molecules. (3) Perhaps most surprising, comparison of mitochondrial gene sequences and the amino acid sequences of the corresponding proteins indicates that the genetic code is different, so that 4 of the 64 codons have different "meanings" from those of the same codons in other genomes (Table 14-4).

The observation that the genetic code is nearly the same in all organisms provides strong evidence that all cells have evolved from a common ancestor. How, then, does one explain the few differences in the genetic code in mitochondria? A hint comes from the recent finding that the mitochondrial genetic code is different in different organisms. Thus UGA, which is a stop codon elsewhere, is read as tryptophan in mitochondria of mammals, fungi, and protozoans but as stop in plant mitochondria. Similarly, the codon AGG normally codes for arginine, but it codes for stop in the mitochondria of mammals and for serine in Drosophila (see Table 14-4). Such variation suggests that a random drift can occur in the genetic code in mitochondria. Presumably, the unusually small number of proteins encoded by the mitochondrial genome makes an occasional change in the meaning of a rare codon tolerable, whereas such a change in a large genome would alter the function of many proteins and thereby destroy the cell.

An im a l Mit o c h o n d ria Co n t a in t h e S im p le s t Ge n e t ic S y s t e m s

Kn o w n 49

Comparisons of DNA sequences in different organisms reveal that the rate of nucleotide substitution during evolution has been 10 times greater in mitochondrial genomes than in nuclear genomes, which presumably is due to a reduced fidelity of mitochondrial DNA replication, DNA repair, or both. Because only about 16,500 DNA nucleotides need to be replicated and expressed as RNAs and proteins in animal cell mitochondria, the error rate per nucleotide copied by DNA replication, maintained by DNA repair, transcribed by RNA polymerase, or translated into protein by mitochondrial ribosomes can be relatively high without damaging one of the relatively few gene products. This could explain why the mechanisms that carry out these processes are relatively simple compared to those used for the same purpose elsewhere in cells. The presence of only 22 tRNAs and the unusually small size of the rRNAs (less than two-thirds the size of the E. coli rRNAs), for example, would be expected to reduce the fidelity of protein synthesis in mitochondria, although this has not yet been tested adequately.

The relatively high rate of evolution of mitochondrial genes makes mitochondrial DNA sequence comparisons especially useful for estimating the dates of relatively recent evolutionary events, such as the steps in primate development.

W h y Are P la n t Mit o c h o n d ria l Ge n o m e s S o La rg e ? 50

Mitochondrial genomes are much larger in plant than in animal cells, and they vary remarkably in their DNA content, ranging from about 150,000 to about 2.5 x 10 6 nucleotide pairs. Yet these genomes seem to encode only a few more proteins than do animal mitochondrial genomes. The

paradox is compounded by the observation that in one family of plants, the cucurbits, mitochondrial genomes vary in size by as much as sevenfold. The green alga Chlamydomonas has a linear mitochondrial genome of only 16,000 nucleotide pairs, the same size as in animals.

Although very little sequence information is available for higher plant mitochondrial DNA molecules, almost all of the 70,000 nucleotide pairs in the large mitochondrial genome of the yeast Saccharomyces cerevisiae have been sequenced, and only about one-third of them code for protein. This finding raises the possibility that much of the extra DNA in yeast mitochondria, and possibly in plant mitochondria as well, is "junk DNA" of little consequence to the organism.

S o m e Org a n e lle Ge n e s Co n t a in I n t ro n s 51

The processing of precursor RNAs plays an important role in the two mitochondrial systems studied in most detail - human and yeast. In human cells both strands of the mitochondrial DNA are transcribed at the same rate from a single promoter region on each strand, producing two different giant RNA molecules, each containing a full-length copy of one DNA strand. Transcription is, therefore, completely symmetric. The transcripts made on one strand - called the heavy strand (H strand) because of its density in CsCl - are extensively processed by nuclease cleavage to yield the two rRNAs, most of the tRNAs, and about 10 poly-A-containing RNAs. In contrast, the light strand (L strand) transcript is processed to produce only eight tRNAs and one small poly-A-containing RNA; the remaining 90% of this transcript apparently contains no useful information (being complementary to coding sequences synthesized on the other strand) and is The processing of precursor RNAs plays an important role in the two mitochondrial systems studied in most detail - human and yeast. In human cells both strands of the mitochondrial DNA are transcribed at the same rate from a single promoter region on each strand, producing two different giant RNA molecules, each containing a full-length copy of one DNA strand. Transcription is, therefore, completely symmetric. The transcripts made on one strand - called the heavy strand (H strand) because of its density in CsCl - are extensively processed by nuclease cleavage to yield the two rRNAs, most of the tRNAs, and about 10 poly-A-containing RNAs. In contrast, the light strand (L strand) transcript is processed to produce only eight tRNAs and one small poly-A-containing RNA; the remaining 90% of this transcript apparently contains no useful information (being complementary to coding sequences synthesized on the other strand) and is

Unlike human mitochondrial genes, some plant and fungal (including yeast) mitochondrial genes contain introns, which must be removed by RNA splicing. Introns have also been found in about

20 plant chloroplast genes. Many of the introns in organelle genes consist of related nucleotide sequences that are capable of splicing themselves out of the RNA transcripts by RNA-mediated catalysis (see p. 109), although these self-splicing reactions are generally aided by proteins. The presence of introns in organelle genes is surprising, as introns are not common in the genes of the bacteria whose ancestors are thought to have given rise to mitochondria and plant chloroplasts.

In yeasts the same mitochondrial gene may have an intron in one strain but not in another. Such "optional introns" seem to be able to move in and out of genomes like transposable elements. On the other hand, introns in other yeast mitochondrial genes have been found in a corresponding position in the mitochondria of Aspergillus and Neurospora, implying that they were inherited from

a common ancestor of these three fungi. It seems likely that the intron sequences themselves are of ancient origin and that, while they have been lost from many bacteria, they have been preferentially retained in those organelle genomes where RNA splicing is regulated to help control gene expression.

Mit o c h o n d ria l Ge n e s Ca n B e D is t in g u is h e d fro m N u c le a r Ge n e s b y Th e ir N o n - Me n d e lia n ( Cy t o p la s m ic ) I n h e rit a n c e