The human mitochondrial genome is only 16.6kb and among the smallest in the animal kingdom. The circular chromosome was completely sequenced by Sanger's team in 1981 and the map is shown below:
The heavy H strand has a higher guanine content, and in this diagram is transcribed in a clockwise direction as a single RNA molecule starting from the PH promoter. The light L strand is transcribed anticlockwise from the PL promoter. Both RNA transcripts are later cleaved to yield functional RNA molecules. Replication of the heavy strand by DNA polymerase commences anticlockwise from the OH replication origin; this eventually exposes the OL origin allowing replication of the light strand to be completed. The red-coloured region near the promoters is known as the D-loop and contains a short length of triple stranded DNA.
The mitochondrial genetic information is very densely packed. There are no introns and only tiny gaps between the genes. [In the diagram above, N1 - N6 are NADH dehydrogenase subunits, cyt b is cytochrome b, OX1 - OX3 are cytochrome oxidase subunits, and the genes for two ATPase subunits partly overlap.] There are 37 mitochondrial genes in total, but only 13 of these code for polypeptides, the remainder being the 2 ribosomal subunits and 22 types of transfer RNA. All the hundreds of other mitochondrial proteins, including DNA polymerase, RNA polymerase, amino acid activating enzymes and all the ribosomal proteins are coded by nuclear genes and imported from the cytosol.
There are about 2000 copies of the mitochondrial genome in a typical cell, so that despite its small size it comprises about 0.5% of the DNA mass. Mitochondrial DNA replication appears to be less accurate than nuclear copying, and it is far from clear how consistency is maintained between these multiple copies, or how their replication is synchronised with the remainder of the cell.
There are substantial differences between the proteins present in mitochondria from different tissues, reflecting the tissue specific patterns of nuclear gene expression. Protein turnover, however, seems to be fairly slow and mitochondrial protein composition does not respond very quickly to dietary or hormonal stimuli. The total number of mitochondria per cell can be changed (for example, through muscle activity) over the course of several weeks.
The import of cytosolic proteins to the mitochondrial matrix space takes place at specialised sites where the inner and outer membranes are in close contact. Cytosolic precursors are marked for import with an N-terminal leader sequence, bearing a substantial positive charge. The leader folds to form an amphipathic alpha helix where the hydrophobic amino acids are concentrated along one face. Receptor proteins in the outer membrane recognise sub-sets of these import signals, and help to direct the precurors towards the import channel. Mitochondrial proteins are usually more basic than their cytosolic counterparts. They probably have a net positive charge at physiological pH, and are strongly influenced by the membrane potential. It appears that the proton motive force provides part of the energy for protein uptake.
Proteins pass through the membranes in an extended linear configuration. It is important that they should not fold fully before import, since the correctly folded enzymes could not pass through the hole. Folding is temporarily prevented in the cytosol by binding to the chaperone protein hsp70, while two further mitochondrial chaperones hsp60 and hsp70 supervise re-folding of the imported proteins after they have entered the matrix space. ATP hydrolysis is required in the cytosol and in the mitochondria for successful import of functional proteins, in addition to the membrane potential and pH gradient. This ATP requirement is probably associated with protein folding.
The matrix targeting sequence is cleaved from the imported proteins by a matrix protease. If the imported protein is destined for the intermembrane space (e.g. myokinase) cleavage of the first signal sequence is believed to expose a second signal directing re-export of the protein through the inner membrane. (The unusually small protein apo-cytochrome c apparently uses the non-specific outer membrane channels to reach the inter-membrane space. Chelation of heme, and consequent adoption of the native protein conformation locks this particular protein into its final position.)