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RNA synthesis in RNA template

 

Viral RNA induces the formation of RNA-dependent RNA polymerase in host cells, which is involved in the replication of viral RNA. Examples are viruses of influenza, rabies, mumps, and measles. RNA replicase constructs complementary RNA strand on the template virus RNA. It serves as a template for RNA synthesis, the same type of initial viral RNA. Both stages are catalyzed by the same enzyme, although each involves different protein factors.

Since the RNA replicase is related only to the virus, obviously, effective antiviral drugs can be developed on this basis.

Protein biosynthesis

 

Protein synthesis is closely connected to the concept of genetic code.

Genetic code is the way to codify amino acid sequence with the help of sequence of nucleotides in all living organisms.

The properties of the genetic code

1. Triple character. The unit of code is a combination of three nucleotides (triplet or codon). All in all there are 64 codons, in other words, each amino acid is codified by three nucleotides in mRNA.

2. Continuity. There are no commas or punctuation signs between the triplets, that is, information is read continuously.

3. Non-overlapping. One and the same nucleotide could not be simultaneously in the structure of two or more triplets.

4. Specificity. A particular codon codes for only one amino acid (AUU is isoleucine, UUU is phenylalanine).

5. Degenerate. One amino acid could correlate to several codons (isoleucine is AUU, AUC, AUA).

6. Universality. Genetic code work is the same in the organism of different structures from viruses to humans.

 

 

Preparatory stage of protein synthesis

20 amino acids, 20 aminoacyl-tRNA-synthetases, more than
20 tRNA, ATP, magnesium ions are necessary.

Amino acid reacts with ATP with the formation of aminoacyl-adenylate, then amino acid is transported from aminoacyladenylate to acceptor stem of tRNA (3’-end):

R–CH(NH2)–COOH + ÀÒP ® R–CH(NH2)–CO~O–ÀÌP + PPi

Aminoacyladenylate

R–CH(NH2)–CO~O–ÀÌP + tRNA ®

® R–CH(NH2)–CO~O–tRNA+ ÀÌP

An enzyme here is aminoacyl-tRNA-synthetase. The presence of magnesium ions is necessary. Aminoacyl-tRNA-synthetases are characterised by absolute specificity. Each amino acid has its own enzyme.

Transfer RNA (tRNA) provides connection between codons of mRNA and amino acids of the future polypeptide chain. There is not less than one tRNA for each amino acid out of 20 amino acids. Each tRNA has about 80 nucleotides. They have the structure of clover leaf in 2-dimensional shape and L-structure in the 3-dimensional shape (Fig. 3).

Fig. 3. The structure of tRNA

The acceptor stem is the site for amino acids binding. It contains CCA sequence.

The TyC (pseudouridilic) loop provides aminoacyl-tRNA binding with ribosome.

The D (dihydrouridilic) loop is a site for the recognition of aminoacyl-tRNA synthetase.

Anticodon loop contains an anticodon which is complimentary to the codon in the mRNA It has 3 nucleotide residues.



A destination of the variable loop is unknown.

3.2.1. Translation

 

Protein biosynthesis is called translation. The sequence of amino acids in the protein is determined by nucleotides sequence of mRNA.

Ribosomesare made up of 2 subunits. They are nucleoproteins which are made up of proteins (65% are at prokaryotes and 50% are at eukaryotes) and ribosomal RNA (rRNA). Ribosomes are characterized by the rate of sedimentation in the centrifugal field in the units proposed by Svedberg: eukaryotes have 80S ribosomes, prokaryotes have 70S ribosomes. Eukaryotes have 60S and 40S ribosomal subunits, prokaryotes have 50S and 30S correspondingly.

There are three stages in translation:

Initiation.

Ribosomal subunits, mRNA, initiation aminoacyl-tRNA (methionyl-tRNA, formyl-methionyl-tRNA), initiation codon in mRNA (AUG), protein initiation factors, cap-recognising factor, GTP and Mg2+ are necessary.

Initiation complex is formed due to binding of protein factors, formyl-methionyl-tRNA and GTP to 30S subunit. mRNA attaches to 30S subunit by AUG codon which is complementary to formylmethionyl-tRNA anticodon. After that protein factor is released. Complex binds 50S subunit which forms active (translating) 70S ribosome. This ribosome has a free aminoacyl site which could react with aminoacyl-tRNA which corresponds to mRNA codon (Fig. 4). The group of ribosomes together with mRNA forms polyribosome or polysome which increases the rate of protein synthesis.

 

Fig. 4. The formation of the translating ribosome

Elongation.

The large ribosomal subunit has 2 sites for tRNA binding: aminoacyl (A) and peptidyl (P). Protein factors also take part in elongation:

Elongation process is subdivided into 3 stages:

1. Codon recognition and aminoacyl-tRNA binding.

2. Peptide bond formation.

3. Translocation.

Stage 1. Aminoacyl-tRNA comes to free aminoacyl centre of ribosome in correspondence to mRNA codon. Elongation factor participates. Energy is derived from GTP. As a result, in the peptidyl site of translating ribosome there is formyl-methionyl-tRNA and in the aminoacyl site there is aminoacyl-tRNA (the first amino acid after methionine).

Stage 2. There is a transfer of methionine residue from methionyl-tRNA to amino group of the new aminoacyl-tRNA. There is transpeptidation reaction. The enzyme here is peptidyltransferase. Methionyl-tRNA is released from peptidyl centre to cytosol. In aminoacyl site dipeptidyl-tRNA is formed and peptidyl centre is free.

Stage 3. Dipeptidyl-tRNA is transferred from aminoacyl centre to peptidyl centre. To do this ribosome moves on one codon relative to mRNA in 5’-3’ direction. Enzyme here is peptidyltranslocase. GTP energy is used. Dipeptidyl-tRNA occupies peptidyl site. Aminoacyl site is free and takes new aminoacyl-tRNA which corresponds to the codon in mRNA. Tripeptidyl-tRNA is formed, and so on.

At each peptide bond synthesis 2 molecules of ATP and 2 molecules of GTP are used.

 

 

Fig. 5. Translation elongation

 

3. Ternimation.

Termination codons (UAA, UAG, UGA), termination factors (release factors) are necessary. Termination codons do not have corresponding tRNA. When termination codon enters ribosome, termination factor binds to it. Specificity of peptidyl-transferase is changed, there is hydrolysis of the bond between synthesized peptide and the last tRNA and protein is released. Energy is derived from GTP.

3.2.2. Post-translational modification of proteins

 

In majority of cases proteins are synthesized in the form of precursors which are biologically inactive molecules. Their functional activity is expressed due to several conversions which are called post-synthetic or post-translational modification (processing).

Examples of post-translational modification of proteins are:

1. Proteolytic cleavage of N-terminal formyl-methionine or methionine.

2. Cleavage of signal peptides.

3. Partial proteolysis.

4. Post-translational modification of proteins on amino acid radical: covalent addition of prosthetic group, methylation of lysine and arginine radicals, acetylation of N-terminal amino acid, phosphorylation of histones and non-histon chromatine proteins, hydroxylation of proline radical, addition of oligosaccharide fragments (glycosylation) to asparagine, serine and threonine radicals, etc.

The formation of the correct proteins structure is done with the help of chaperones. Hydrophobic parts at the surface of the chaperones-70 globule interact with hydrophobic parts of the synthesized chain protecting it from wrong interactions with other proteins in the cytosol. Chaperones-60 take part in the correction of spatial structure of wrongly folded or damaged chain.

Mutations in the chaperone, which is the part of the eye lens lead to the opacity of the lens due to protein aggregation and the development of cataracts.

 


Date: 2016-04-22; view: 957


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