Protein synthesis

Cells consist mainly of proteins, which constitute more than half of their dry weight.

Proteins determine shape and structure of the cell and are important for its

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maintenance, growth and development. The proteins are made from an assortment of 20 different amino acids, each with a distinct chemical property (ALBERTS et al.

1994c). Protein synthesis is a complex process which requires a series of preparatory steps. First, a molecule of mRNA must be copied from DNA that encodes the protein through a specific transcription factor and RNA polymerase II. Meanwhile, in the cytoplasm, each of the 20 amino acids from which the protein is to be synthesized must be pre-attached to its specific tRNA molecule and the subunits of the ribosome where the new protein is to be made are preloaded with translation initiation factors (ALBERTS et al. 1994a). Protein synthesis begins when all components come together in the cytoplasm to form a functional ribosome. The sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids to produce a distinctive protein chain, as specified by the DNA sequence of its gene (SOLOMON et al. 1996c).

Translation is the process by which the mRNA sequence is read and translated into an amino acid polymer (protein) via the ribosomes which are conformed by two different subunits, each made up of both ribosomal RNA and more than 50 different proteins. In eukaryotic cells, initiation of mRNA translation into a protein follows a sequence of well defined reactions that require specific proteins termed eukaryotic translation initiation factors (eIFs) [Fig. 12]. The first step involves the binding of guanosine triphosphate (GTP) and the initiator complex methionine-tRNA (Met-tRNA) to eukaryotic initiation factor-2 (eIF2) to form the Met-tRNA-eIF2-GTP (ternary complex), which is then transferred to the 40S ribosomal subunit in a reaction catalyzed by eukaryotic initiation factor 1A (eIF1A) where it is stabilized by binding of the multi-subunit initiation factor 3 (eIF3) to form the 40S preinitiation complex (40S-eIF3-Met-tRNA-eIF2-GTP). The 40S preinitiation complex then binds to the capped 5’

end of an mRNA transcript in a reaction requiring eIF4F, eIF4A, and eIF4B and progresses until it reaches the appropriate initiation codon (AUG) where the 40S complex is bound to mRNA through codon-anticodon base pairing to form the 40S initiation complex (40S-mRNA-eIF3-Met-tRNA-eIF2-GTP). Initiation factor eIF5 interacts with the 40S initiation complex to produce hydrolysis of the 40S subunit-bound GTP. This hydrolysis leads to the release of the initiation factors subunit-bound to the

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complex and to the concomitant joining of the 60S ribosomal subunit to the 40S complex to form the functional 80S initiation complex (CHAUDHURI et al. 1997;

CHAUDHURI et al. 1999).

A group of three RNA nucleotides in the mRNA called a codon is the signal to add a specific amino acid. Translation normally begins at the first AUG sequence after the 5’cap. The first amino acid added is methionine (because AUG codes for methionine) and forms the amino terminal (N terminal) end for new protein (ALBERTS et al.

1994c).

The addition of other amino acids to the growing polypeptide is called elongation (Fig. 13). For each codon, a new amino acid must be transported to the ribosome by the transfer RNA (tRNA). As mentioned above, the complete set of tRNA molecules is produced within the nucleus by RNA polymerase III. The tRNA recognizes both an amino acid and a group of three nucleotides to transfer this specific amino acid into a place in the ribosome where it is added to the growing protein polymer. Amino acids are covalently linked to their respective tRNA molecule by aminoacyl-tRNA synthetases. The resulting complexes, called aminoacyl-tRNAs bind to the mRNA coding sequence such that the amino acids are aligned in the correct order to form the polypeptide chain (SOLOMON et al. 1996c). The mRNA sequence eventually contains a signal to stop with one of three sequences UAA, UAG or UGA. This prevents further addition of amino acids and produces the C terminal end of the protein (ALBERTS et al. 1994a; ALBERTS et al. 1994c).

Oocyte maturation and development of preimplantation embryos have a profound need for synthesis of proteins for both housekeeping functions and cell specialization (HYTTEL et al. 2000b; SIRARD et al. 1989). In bovine oocytes protein synthesis and protein phosphorylation increase following GVBD and pronucleus formation (CHIAN et al. 1999; CHIAN et al. 2003; KASTROP et al. 1991a).

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Fig: 12. Initiation of transcription in eukaryotic cells.

a) Dissociation of the ribosomal subunits b) Binding of GTP and Met-tRNAto eIF2 to form the Met-tRNA-eIF2-GTP (ternary complex). This complex is then transferred to the 40S ribosomal subunit in a reaction catalyzed by eIF1A and stabilized by the binding of eIF3 to form the 40S preinitiation complex (40S-eIF3-Met-tRNA-eIF2-GTP). Then this complex binds to the capped 5’ end of mRNA in a reaction requiring eIF4F, eIF4A, and eIF4B. c) Initiation factor eIF5 interacts with the 40S initiation complex resulting in hydrolysis of 40S subunit-bound GTP. This hydrolysis leads to the release of initiation factors subunit-bound to the complex and the concomitant joining of the 60S ribosomal subunit to the 40S complex to form the functional 80S initiation complex. A ribosome contains two binding sites for tRNAs that recognize the adjacent codons: The A site (aminoacyl-tRNA site) and the P site (peptidyl-tRNA site). Adapted from SOLOMON et al. 1996c with data obtained by CHAUDHURI et al.

1997.

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Fig: 13. Elongation process of the polypeptide chain in the protein synthesis.

The mRNA passes through a groove formed between the two ribosomal subunits. The A site (aminoacyl-tRNA site) binds an aminoacyl-tRNA that will be used to add a amino acid to the growing chain and the P site (peptidyl-tRNA site) binds the tRNA that is linked to the growing polypeptide chain. Adapted from SOLOMON et al. 1996c.

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2.4.3 Gene expression in preimplantation embryos