Surprises from Mitochondrial Ribosome Research

Surprise 1

Mammalian mitochondrial ribosomes, the first to be discovered 1,2, are 55S ribosomes!

The prevailing notion was that ribosomes come in two forms, the smaller, 70S variety in prokaryotes and the larger, 80S kind in eukaryotes.  It was expected that organellar ribosomes would be 70S particles (reminiscent of their ancestral prokaryotic origins) as reported later for Neurospora mitochondrial ribosomes.

1. O’Brien, T.W. and Kalf, G.F., Ribosomes from Rat Liver Mitochondria. II. Partial Characterization. J. Biol. Chem., 242:2180 (1967).

2. O’Brien, T.W., The General Occurrence of 55S Ribosomes in Mammalian Liver Mitochondria. J. Biol. Chem., 245:3409 (1971).

Is the slower 55S ribosome in mammalian mitochondria

(A) a degraded ribosome?

(B) a subribosomal particle?

(C) a complete “mini-ribosome”?

 Surprise 2

(D) None of the above.

The 55S mitoribosomes of mammals are LARGER than bacterial 70S ribosomes!

Their mass is greater3 and so also are their physical dimensions.

3. Hamilton, M.G. and O’Brien, T.W.,Ultracentrifugal Characterization of the Mitochondrial Ribosome and Subribosomal Particles of Bovine Liver: Molecular Size and Composition. Biochemistry, 13:5400 (1974).

 Surprise 3

While they are larger than bacterial ribosomes, 55S mitoribosomes actually contain smallerRNAs!

So small, that some investigators once thought they were too small to be ribosomal RNAs4.

4. Attardi B, Attardi G., Sedimentation properties of RNA species homologous to mitochondrial DNA in HeLa cells. Nature 224:1079-1083 (1969)

With their smaller RNAs, 55S mitoribosomes are only about 25% RNA, compared to 65%, for 70S bacterial ribosomes.

Surprise 4

The 55S mitoribosomes contain many more proteins5 than do the 70S bacterial ribosomes!

5. Matthews, D.E., Hessler, R.A., Denslow,N.D., Edwards, J. and O’Brien, T.W., Protein Composition of Bovine Mitochondrial Ribosomes. J. Biol. Chem., 257: 8788-8794 (1982).

They contain as many as 85 proteins, compared to only 53 proteins in the E. coli ribosome.

With smaller RNAs and increased protein content, the buoyant density of 55S mitoribosomes is much less than that of the bacterial ribosomes, a major factor contributing to their slower sedimentation rate.

What are all of the extra proteins doing in mammalian mitoribosomes, and where did they come from?

Surprise 5

The nucleotide sequence of mammalian mitoribosomal RNAs was known6 before the sequence of any cytoplasmic ribosomal RNA was determined.

6. Anderson, S. et al., Sequence and Organization of the Human Mitochondrial Genome, Nature, 290:457-465 (1981).

Surprise 6

The 55S mitoribosomes are products of two different genomes; their rRNA is encoded in mitochondrial DNA6, while all of their proteins are encoded by nuclear genes. All of the proteins in 55S mitoribosomes are synthesized on extramitochondrial ribosomes in the cytoplasmic compartment7,8 , from which they must be imported.

7. Schieber, G.L. and O’Brien, T.W.,Cytoplasmic Synthesis of Proteins of the Mammalian Mitochondrial Ribosome. In: Nucleo-Mitochondrial Interactions, R. Schweyen and F. Kaudewitz (eds.), De Gruyter Press (1983), pp.469-479.

8. Schieber, G.L. and O’Brien, T.W., Site of Synthesis of the Proteins of Mammalian Mitochondrial Ribosomes: Evidence from Cultured Bovine Cells. J. Biol. Chem., 260:6367-6372 (1985).

Where are the genes for the different mitoribosomal proteins located?

On which chromosomes?

Are the genes for mitoribosomal proteins clustered, as in prokaryotic ribosomal operons, or are they dispersed?

How is transcription of mitoribosomal genes regulated?

How is the nuclear transcription of mitoribosomal genes coordinately regulated with the mitochondrial transcription of rRNA for mitoribosome assembly?

Surprise 7

Mitochondrial ribosomes in different organisms are more diverse9 in terms of their composition and physical-chemical properties than bacterial or eukaryotic cytoplasmic ribosomes!

9. O’Brien, T.W., and Matthews, D.E., Mitochondrial Ribosomes. In: Handbook of Genetics, Vol. 5, Robert C. King (ed.), Plenum Publishing Co., 1976, pp. 535-80.

Surprise 8

While mammalian mitoribosomes resemble bacterial ribosomes more so than eukaryotic cytoplasmic ribosomes, they are distinguished from both kinds of ribosomes on the basis of their antibiotic susceptibility10,11.

10. Denslow, N.D. and O’Brien, T.W.,Susceptibility of 55S Mitochondrial Ribosomes to Antibiotics Inhibitory to Prokaryotic Ribosomes: Lincomycin, Chloramphenicol, and PA114A. Biochem. Biophys. Res. Commun., 57:9 (1974).

11. Denslow, N.D. and O’Brien, T.W., Antibiotic Susceptibility of the Peptidyl Transferase Locus of Bovine Mitochondrial Ribosomes. Eur. J. Biochem., 91:444-448(1978).

Mammalian mitoribosomes thus have a distinctive antibiotic susceptibility. Some of the adverse side effects of therapeutically important antibacterial antibiotics are ascribed to their inhibitory effects on mitoribosomes (Chloramphenicol induced aplastic anemia; aminoglycoside ototoxicity, and others).

Surprise 9

Although the genes for proteins of mammalian mitochondrial and cytoplasmic ribosomes are all located in the nucleus, the genes for mitoribosomal proteins are evolving at faster rates12,13.

12. Matthews, D.E., Hessler, R.A. and O’Brien,T.W., Rapid Evolutionary Divergence of Proteins in Mammalian Mitochondrial Ribosomes. FEBS Letters, 86:76(1978).

13. Pietromonaco, S., Hessler, R.A. and O’Brien, T.W., Evolution of Proteins in Mammalian Cytoplasmic and Mitochondrial Ribosomes. J. Mol. Evolution, 24:110-117 (1986).

Are all of the mitoribosomal proteins evolving rapidly?

Or is it only the rRNA binding proteins14 which co-evolve with the rapidly evolving mitoribosomal RNAs?

14. Piatyszek, M., Denslow, N.D. and O’Brien,T.W., RNA Binding Proteins of the Large Subunit of Bovine Mitochondrial Ribosomes, Nucleic Acid Research, 16: 2565-2583 (1988).

Surprise 10

Mammalian mitoribosomes bind GTP with high affinity, unlike other kinds of ribosomes15.

15. Denslow, N.D., Anders, J.C., and O’Brien,T.W., Bovine Mitochondrial Ribosomes Possess a High Affinity Binding Site for Guanine Nucleotides, J. Biol. Chem., 266:9586-9590(1991).

The intrinsic GTP binding site has been identified as the small subunit protein, S5 in the bovine mitoribosome.

Liu, J., Anders, J.C., and O’Brien, T.W. Purification, Sequence Analysis and Cloning of cDNA for the GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes, Ninth Symposium of the Protein Society, Boston, July 8-12, 1995.

 Surprise 11

Mammalian mitoribosomal protein S5 is unique; no homologues exist in bacterial or cytoplasmic ribosomes!

What is the function of the GTP binding protein S5?

Liu, J., Anders, J. C. and O’Brien, T. W.GTP Binding Protein in the Small Subunit of Bovine Mitochondrial Ribosomes: a Protein in Search of Function. Cold Spring Harbor Meeting on Translational Control, September 4 – 8, 1996.

Surprise 12

Mammalian mitochondrial mRNA molecules lack 5′ untranslated regions and have no known sequence or structural features, such as 5′ caps or Shine-Dalgarno sequences which promote mRNA binding to eukaryotic or bacterial ribosomes, respectively. Yet, mammalian mitoribosomes have a high intrinsic affinity for mitochondrial mRNA16.

What features of mitochondrial mRNA determine its interaction with mitoribosomes?

16. Denslow, N.D., O’Brien, T.W., Michaels,G.S., Montoya, J. and Attardi, G., Mechanism of mRNA Binding to Bovine Mitochondrial Ribosomes, J. Biol. Chem., 264: 8328-8338 (1989).

Surprise 13

Some mammalian mitochondrial mRNA molecules are bicistronic, coding for two proteins, in overlapping/out of frame open reading frames6.

How do mammalian mitoribosomes initiate protein synthesis on such bicistronic mRNAs?

What regulates the translation frequency of each of the overlapping reading frames?

6. Anderson, S. et al., Sequence and Organization of the Human Mitochondrial Genome, Nature, 290:457-465 (1981).

What Surprises Lie Ahead?