PROTEIN SYNTHESIS
CENTRAL DOGMAThe genetic code is a dictionary that identifies the correspondence between a sequence of nucleotide bases and a sequence of amino acids. Each individual word in the code is composed of three nucleotide bases. These genetic words are called codons. Codons are presented in the messenger RNA (mRNA) language of adenine (A), guanine (G), cytosine (C), and uracil (U). Their nucleotide sequences are always written from the 5′-end to the 3′-end. The four nucleotide bases are used to produce the three-base codons. There are, therefore, 64 different combinations of bases, taken three at a time. The Genetic Code
Any codon sequence when translated, it gives an amino acid to the growing polypeptide chain during protein synthesis (Translation process). e.g.: 5′-AUG-3′ codes for methionine. Termination (“stop” or “nonsense”) codons: Three of the codons, UAG, UGA, and UAA, do not code for amino acids, but rather are termination codons. When one of these codons appears in an mRNA sequence, it signals termination of protein synthesis..
Characteristics of the genetic code
1.Specificity: The genetic code is specific (unambiguous), that is, a particular codon always codes for the same amino acid. 2.Universality: The genetic code is virtually universal, i.e the specificity of the genetic code has been conserved from very early stages of evolution. 3.Degeneracy: The genetic code is degenerate (sometimes called redundant). Although each codon corresponds to a single amino acid, a given amino acid may have more than one triplet coding for it. e.g. arginine is specified by six different codons.4. Nonoverlapping and commaless: The genetic code is nonoverlapping and commaless, that is, the code is read from a fixed starting point as a continuous sequence of bases, taken three at a time. e.g. ABCDEFGHIJKL is read as ABC/DEF/GHI/JKL without any “punctuation” between the codons.
Components Required for Translation A large number of components are required for the synthesis of a protein. These include all the amino acids that are found in the finished product, the mRNA to be translated, transfer RNA (tRNA), functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain (so called initiation factors, elongation factors and termination factors). 1. Amino acids All the amino acids must be available at time of protein synthesis. If one amino acid is missing (for example, if the diet does not contain an essential amino acid), translation stops at the codon specifying that amino acid producing an aberrant or truncated protein.
2. Transfer RNA In humans, there are at least 50 species of tRNA, whereas bacteria contain 30-40 species. Each a.a. has at least 1 t-RNA. Those a.a(s) encoded by more than one codon have more than one specific t-RNA. Each tRNA molecule has an attachment site for a specific (cognate) amino acid at its 3′-end. The carboxyl group of the amino acid is in an ester linkage with the 3′-hydroxyl of the ribose moiety of the adenosine nucleotide in the —CCA sequence at the 3′-end of the tRNA. [Note: When a tRNA has a covalently attached amino acid, it is said to be charged; when tRNA is not bound to an amino acid, it is described as being uncharged. The amino acid that is attached to the tRNA molecule is said to be activated].
Each tRNA molecule also contains a three-base nucleotide sequence —anticodon—that recognizes a specific codon on the mRNA. This codon will specify the insertion of the amino acid carried by that tRNA into the growing peptide chain. The attachment of an a.a. to its specific t-RNA requires the activity of a family of enzymes called aminoacyl-tRNA synthetases with the help of one molecule of ATP. The resulting amino acid- tRNA complex is called [aminoacyl t-RNA]
3.Messenger RNA Specific mRNA must be present as a template for the synthesis of the desired polypeptide chain. [Note: Interactions between certain regulatory proteins that bind the 5′-cap and the 3′-tail of eukaryotic mRNA mediate circularization of the mRNA and likely prevent the use of incomplete mRNA in translation.]4. Functionally competent ribosomes Ribosomes are large factories for protein synthesis. They are formed by proteins and ribosomal RNA (rRNA). They consist of two subunits—one large and one small—whose relative sizes are generally given in terms of their sedimentation coefficients, or S (Svedberg) values.
The prokaryotic 70S ribosome are consisted of both 50S and 30S ribosomal subunits while the eukaryotic 60S and 40S subunits form an 80S ribosome. The large ribosomal subunit catalyzes formation of the peptide bonds that link amino acid residues in a protein. The small subunit binds mRNA and is responsible for the accuracy of translation by ensuring correct base-pairing between the codon in the mRNA and the anticodon of the tRNA.
Ribosomal proteins are present in considerably greater numbers in eukaryotic ribosomes than in prokaryotic ribosomes. They play very important roles not only structural but also in the interactions with other components of the translation. The ribosome has three binding sites for tRNA—A, P, and E sites—each of which extends over both ribosomal subunits. Together, they cover three neighboring codons.
During translation, the A site binds an incoming aminoacyl-tRNA as directed by the codon currently occupying this site. This codon specifies the next amino acid to be added to the growing peptide chain. The P-site codon is occupied by peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized. The E site is occupied by the empty tRNA as it is about to exit the ribosome.
5. Protein factors Initiation, elongation, and termination (or release) factors are required for peptide synthesis. Some of these protein factors perform a catalytic function, whereas others appear to stabilize the synthetic machinery.6. ATP and GTP are required as sources of energy In eukaryotes, 7 high energy bonds (ATP and GTP) are required for the translation process. One ATP and one GTP are needed for the initiation and one GTP for termination (Release). However, cleavage of four high-energy bonds is required for the addition of one amino acid to the growing polypeptide chain: two from ATP in the aminoacyl-tRNA synthetase and two from GTP—one for binding the aminoacyl-tRNA to the A site and one for the translocation step.
Steps in Protein Synthesis
The mRNA is translated from its 5′-end to its 3′-end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNA often have several coding regions, each has an initiation region and a termination region i.e they are polycistronic, while eukaryotic mRNA has only one coding region, that is, it is monocistronic. Initiation: It involves the assembly of the components of the translation machine before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP and ATP, and initiation factors that facilitate the assembly of this initiation complex (in prokaryotes:IF1, IF2,IF3, while over 10 in eukaryotes referred as “eIFs”).How can ribosomes identify or recognize the nucleotide sequences that initiate translation?
1. In eukaryotes, the 40S ribosomal subunit (aided by members of the elF-4) binds to the 5′-end cap of the mRNA and moves down the mRNA until it encounters the “initiator codon AUG”. This “scanning” process requires ATP. 2. The “Initiation codon” AUG is recognized by a special initiator tRNA. Recognition is aided by IF-2 bound to GTP in prokaryotes and eIF2-GTP (plus additional eIF) in eukaryotes. The initiator tRNA enters the ribosomal P site charged with methionine and goes directly to the P site.[Note: The initiator tRNA is the only tRNA recognized that goes directly to the P site carrying the N- terminal methionine; which is usually removed before completion of translation].
B. Elongation
Elongation of the polypeptide chain involves the addition of a new a.a to the carboxyl end of the growing chain. During elongation, the ribosome moves from the 5′-end to the 3′-end of the mRNA that is being translated. Once the A site of the ribosome is on the next codon template, the next coming appropriate (aminoacyl t-RNA) will arrive and being delivered by the help of elongation factors (Efs). [Note: In eukaryotes, elongation factors are EF-1α and EF-1βγ]. The formation of the peptide bonds is catalyzed by peptidyltransferase, whose activity in prokaryotes is intrinsic to the 23S rRNA found in the 50S ribosomal subunit (so that called ribozyme).Each time of elongation, the ribosome advances three nucleotides toward the 3′-end of the mRNA. This process is known as translocation.This utilizes EF-2 in eukaryotic cells and GTP; while in prokaryotes, requires the participation of EF-G (eukaryotic cells use EF-2) and GTP hydrolysis. This step causes movement of the uncharged tRNA (i.e empty tRNA) into the ribosomal E site (before being released), and movement of the peptidyl-tRNA into the P site.
C. Termination (Release): It occurs when one of the three termination codons moves into the A site. In eukaryotes a single release factor, eRF, recognizes all the termination codons. Once this happens, it induces peptidyltransferase enzyme to hydrolyze the bond linking the peptide chain to the t-RNA at the A site with the aid of one GTP hydrolysis. This causes the nascent protein to be released from the ribosome. The ribosomal subunits, m- RNA, t-RNA, and protein factors can be recycled and used to synthesize another protein.
Polysomes Translation begins at the 5′-end of the mRNA, with the ribosome proceeding along the RNA molecule. Because of the length of most mRNAs, more than one ribosome at a time can generally translate a message. A complex of one mRNA and a number of ribosomes is called a polysome or polyribosome.
Consequences of Altering the Nucleotide Sequence (Mutations)الطفرات الوراثية، عواقبها و علاجاتها
Mutations are alterations in the normal nucleotides sequences
1. Point mutation: i.e changing a single nucleotide base on m-RNA chain, it can lead to any of the following 3 results: A. Silent mutation: The codon containing the changed base may code for the same amino acid. e.g, if the serine codon UCA is given a different third base—U—to become UCU, it still codes for serine. B. Missense mutation: The codon containing the changed base may code for a different amino acid. e.g, if the serine codon UCA is given a different first base—C—to become CCA, it will code for a different amino acid, in this case, proline. C. Nonsense mutation: The codon containing the changed base may become a termination codon. e.g, if the serine codon UCA is given a different second base—A—to become UAA, the new codon causes termination of translation at that point, and the production of a shortened (truncated) protein.2. Other mutations: Trinucleotide repeat expansion: a sequence of three bases that is repeated in tandem will become amplified in number, so that too many copies of the triplet occur. If this occurs within the coding region of a gene, the protein will contain many extra copies of one amino acid. e.g, amplification of the CAG codon leads to the insertion of many extra glutamine residues in the huntington protein, causing the neurodegenerative disorder, Huntington disease.
If the trinucleotide repeat expansion occurs in the untranslated portion of a gene, the result can be a decrease in the amount of protein produced as seen, for example, in fragile X syndrome and myotonic dystrophy.
b. Splice site mutations: Mutations at splice sites can alter the way in which introns are removed from pre-mRNA molecules, producing aberrant proteins. c. Frame-shift mutations: One or two nucleotides are either deleted from or added to the coding region of a message sequence leading to alter the reading frame. This can result in an absolutely different protein or producing a termination codon and ending with a truncated protein.
Sometimes, 3 nucleotides can be added or deleted resulting in no change in frame reading but in only addition or deletion of one amino acid.