Protein Biosynthesis

Defects in Mitochondrial Oxidative Phosphorylation and Disease: Deficiency in Mitochondrial Translation

There are 13 proteins encoded by the mitochondrial genome (mitochondrial DNA) that comprise the respiratory chain (Fig. 14.19) located in the inner mitochondrial membrane. About 150 different proteins are required for the translation of these 13 respiratory chain proteins. Translation deficiencies caused by nuclear gene mutations generate mutations in transfer RNA (tRNA), ribosomal RNA (rRNA), and the proteins involved in translation. Early on, some of these were identified as mutations in tRNA-modifying enzymes, proteins of the ribosome, aminoacyl-tRNA synthases, as well as elongation and termination factors, and these mutations lead to a variety of diseases. The nuclear DNA–derived proteins are imported from the cytoplasm into the mitochondrion (see Fig. 11.19 ). The diseases caused by mutations in nuclear genes that are involved in translation or in the regulation of translation are shown in Table 11.1 .

Nuclear genes encode most of the mitochondrial proteins that number as many as 1400 proteins. Of these, about 150 different nuclear-derived proteins take part in the translation of the 13 proteins encoded by the mitochondrial genome. These mitochondrial genes encode proteins that are involved in the assembly of the components of oxidative phosphorylation. The nuclear-derived proteins also are involved in many other mitochondrial functions, including involvement in DNA replication, metabolism, maintenance, transcription, repair, and dNTP (deoxytrinucleotide) syntheses.

Protein Synthesis in the Mitochondrion

There are two differences in the mitochondrial genetic code from the nuclear genetic code: in the nuclear genetic code, UGA is a stop codon , whereas in the mitochondrial genetic code UGA codes for tryptophan ; AUA codes for isoleucine in the nuclear genetic code, whereas AUA codes for methionine in the mitochondrial genetic code. UAG codes for a stop codon in both systems and the other codons are similar (see Appendix 2 ). Although nuclear DNA contains many tRNA genes, there are only 22 tRNA genes in the mitochondrion that are needed to read all the codons using a unique mitochondrial mechanism . In addition, the mRNAs (messenger RNAs) contain few nucleotides in the 5′-end untranslated regions and cap structure.

Mitochondrial DNA encodes proteins (through mitochondrial mRNA) all of which are hydrophobic and located in the inner mitochondrial membrane. The translation of mitochondrial mRNA occurs in a complex bound to the inner membrane through electrostatic forces and protein interactions. One such protein is LETM1, an inner membrane protein, which may serve as an anchor protein in the formation of a complex with the mitochondrial ribosome.

Mutations can occur in any component of the translation machinery to produce deficiencies in protein synthesis presenting in any mechanism of inheritance, although the mode of inheritance of mitochondrial genes is maternal . These mutations generate a deficiency in oxidative phosphorylation affecting any of the complexes in the respiratory chain containing the proteins of mitochondrial derivation; apparently, only complex II is spared. Mitochondrial-derived diseases are now a rapidly growing area of genetic diseases in medicine.

Mitochondrial Encephalomyopathy With Lactic Acidosis and Stroke-Like Episodes

Mitochondrial encephalomyopathy with lactic acid and stroke-like episodes (MELAS) is one of the prominent diseases in this category. It is caused by a mutation in any of five mitochondrial genes (MTND1, MTND5, MTTH, MTTV, and especially MTTL1). Mutation in the MTTL1 gene causes more than 80% of all the cases of MELAS. The MTTL1 mutation involves a tRNA, the mutation being A3243G (adenine 3243 to guanine) in the tRNA ^{leu (UUR)} gene. MELAS is relatively rare, occurring in the range of 1 person in 4000. In Fig. 11.1A the mitochondrial genome emphasizing the gene mutations causing various mitochondrial genetic diseases (MELAS is located on the upper right where the mutation in leu tRNA gene is adenine to guanine at nucleotide 3243) is shown. Fig. 11.1B is a listing of aspects of the diagnosis of MELAS.

The structural changes in mitochondrial tRNAs that lead to disease are shown in two examples in Fig. 11.2 .

In Table 11.2 are listed mitochondrial tRNA genes that are mutated in disease.

In early childhood (and also in older patients), MELAS patients have seizures, recurrent headaches, vomiting, anorexia (low body weight and fear of gaining weight), intolerance of exercise, and weakness in arms and legs. Stroke-like episodes are common, and they progressively damage motor functions, vision (sometimes resulting in blindness), hearing, and mental ability. MELAS is caused primarily by defects in oxidative phosphorylation through damage to complex I and to complex IV. Precisely 8% of MELAS patients harbor the A3243G mutation in the Mt-tRNA ^Leu gene wobble position ( Fig. 11.2 ). This is an active subject of new research and vital to the diagnosis of new diseases spawned by mutations affecting translation in the mitochondria.

Protein Synthesis Directed by the Nucleus

The sequence of events leading to the formation of a protein takes place first in the nucleus and then in the cytoplasm. The major steps are: transcription , translation , and posttranslational modifications . The process of transcription of a specific gene takes place in the nucleus (see Chapter 12: Transcription). Genetic information is first encoded in an mRNA molecule that is exported to the cytoplasm at the ribosome where the new protein molecule is synthesized. mRNA contains three bases coding for a specific amino acid. The amino acid is ferried to the codon site when the anticodon contained in a tRNA interacts with the codon of the message. The first amino acid to attach is signaled by an initiation codon . Each amino acid is annealed to the previous one in a peptide bond. The process ( elongation ) continues until the protein has been completed (signaled by a stop codon ). The fully formed polypeptide chain is released and is folded into the tertiary structure. Most of these events are summarized in simplified form in Fig. 11.3 .

The manner in which the amino acids are recognized through the codons on the mRNA and the interaction with the anticodons on the tRNAs is shown in Fig. 11.4 .

mRNA is transcribed from DNA where the base pairing partners are G–C and A–T(U). Guanine (G) pairs with cytosine (C), and adenine (A) pairs with thymine (T) in DNA or with uracil (U) in RNA. Consequently, during transcription, a G in DNA will transcribe for a C in mRNA, and a C in DNA will transcribe for a G in mRNA. An A in DNA will transcribe for a U in mRNA, and a T in DNA will transcribe for an A in mRNA. In Table 11.3 the triplet bases are listed that code for the amino acids.

The code is degenerated in many cases such that four different triplets in mRNA can code for the same amino acid (e.g., valine) . In many of these cases the first two bases are identical but the third base varies.

The Ribosome

The mammalian ribosome consists of two subunits. The large subunit has a sedimentation value of 60S (determined in an analytical ultracentrifuge or by sucrose density gradient centrifugation) equivalent to about 2.8 million Daltons and contains as many as 49 proteins and 4880 nucleotides in the form of 28S, 5.8S, and 5S rRNAs. The small subunit is 40S, weighs 1.4 million Daltons, and contains 33 proteins and 1874 nucleotides in the form of 18S rRNA . The RNAs can account for half the weight of the ribosome. The complex of both subunits has a sedimentation constant of 80S and weighs 14.22 million Daltons. The nucleolus is the site of rRNA production ( Fig. 11.5 ). The ribosomal proteins , synthesized in the cytoplasm, are transported into the nucleolus and the nucleoplasm. Four of the rRNAs are synthesized in the nucleolus ( Fig. 11.5 ).

The ribosomal subunits are assembled in the nucleoplasm and transported individually out of the nucleus (through the nucleopore) to the cytoplasm where the ribosomal dimer is found either freely in the cytoplasm or on the rough endoplasmic reticulum (RER). Free ribosomes may be in the form of polyribosomes . The 80S ribosome is pictured in Fig. 11.6 .

The ribosomal subunits combine in the cytoplasm. Free ribosomes in the cytoplasm make proteins that remain in the cytoplasm. The ribosomes that are located within the mitochondria resemble bacterial (prokaryotic, without a defined nucleus) ribosomes at 70S that are smaller than eukaryotic (with a defined nucleus) ribosomes (80S). A human cell may contain a few million ribosomes, and cells that are active in protein synthesis would be expected to have more ribosomes than those less active in protein synthesis.

Ribosomes read mRNA from the 5′-end to the 3′-end. Translation can occur either on free polysomes or polysomes associated with a number of cytoplasmic structures, including the endoplasmic reticulum (ER), where proteins destined for the export of posttranslational modification are synthesized. The polypeptide elongates, forms its tertiary structure, and is released into the ER and then to the Golgi apparatus where it can be designed for destination to a secretory granule (to be transported to the extracellular space), to the lysosome , where it can be broken down or to the plasma membrane. The anatomy of a functional ribosome is shown in Fig. 11.7 . There are three important sites on the ribosome, the A site, the P site, and the E site. The A site is the aminoacyl acceptor site, the P site is the peptidyl site, and the E site is the location of the exit. The reading of mRNA begins with a start codon , AUG , and terminates with a stop codon , UAG ( Table 11.3 ). The first amino acid added is methionine for the AUG codon. It is transferred to the P site on the large subunit being brought to that site by the methionine tRNA which has the anticodon, UAC, that forms a complex with mRNA at the AUG site ( Fig. 11.8 ). This is the start of the growing peptide.