Gene expression Translation

The characteristics of the genetic code The genetic code is a set of rules defining how the four-letter code of DNA (RNA) is translated into the 20-letter code of amino acids, which are the building blocks of proteins.  The characteristics of the genetic code Triplet code Degenerate (redundant) Unambiguous Nonoverlapping Comma less Universal

Genetic code table

Translation Translation is the synthesis of proteins directed by a mRNA template. Principle: a template is required Conditions: mRNA Ribosomes Amino acids tRNAs Enzymes, translation factors Energy (ATP, GTP)

Aminoacyl-tRNA synthesis

a ribosome

Initiation of translation (prokaryotic cells) fmet-tRNA formation 30 S + IF3 → dissociation of the ribosome + mRNA fmet-tRNA is brought to start codon AUG (IF2/GTP → IF2/GDP) fmet-tRNA recognizes AUG: the anticodon UAC (fmet-тРНК) base-pairs with a complementary codon AUG on mRNA +IF1 which binds A site and blocks it + 50 S → 70 S (reassociation of the ribosome) Dissociation of initiation factors (IF1,2,3) fmet-tRNA is at Р site of the ribosome, А site is empty

The selection of an initiation site (AUG codon) depends on the interaction between the 30S subunit and the mRNA template. Shine-Dalgarno (SD) sequence (AGGAGG) is a ribosomal binding site in prokaryotic mRNA, generally located around 8 bases upstream of the start codon AUG. SD is complementary to a pyrimidine-rich sequence at 3’end of 16 S rRNA in a small subunit.

Elongation of translation (prokaryotic cells) Formation of the next aminoacyl-tRNA The aminoacyl-tRNA is brought into A site of the ribosome (Tu/GTP → Tu/GDP) Codon recognition: the mRNA codon in the A site of the ribosome forms hydrogen bonds with the anticodon of an entering tRNA Peptide bond formation: an rRNA molecule catalyzes the formation of a peptide bond between the amino acid in the P site with the new amino acid in the A site. (23 S rRNA in the 50S ribosomal subunit has peptidyl transferase activity) Translocation of the ribosome by one codon (G/GTP → G/GDP) Regeneration of Tu/GTP: Tu/GDP + Ts → Tu/Ts + GDP Tu/Ts + GTP → Tu/GTP + Ts

Termination of translation (prokaryotic cells) Stop codons: UAA, UAG, UGA Release factors: RF1, RF2, RF3, RRF RF1 recognizes UAG and UAA RF2 recognizes UGA and UAA These factors trigger the hydrolysis of the bond in peptidyl-tRNA and the release of the newly synthesized protein from the ribosome. RF3 facilitates binding of RF-1 or RF-2 to the ribosome and their release. It has GTPase activity. RRF (ribosomal recycling factor) is required for release of uncharged tRNA from the P site, and dissociation of the ribosome from mRNA with separation of the two ribosomal subunits.

a polysome Several ribosomes can translate an mRNA at the same time, forming a polysome (a polyribosome).

Translation properties In eukaryotic cells 80S (40S, 60S) methionine (met) eIF 1, 2, 3, 4A, 4B, 4C, 4Е, 5 etc. (12) Cap (the scanning hypothesis); Kozak sequence; an internal ribosome entry site in the 5'UTR 40S + met-tRNA + mRNA in prokaryotic cells Ribosomes 70S (30S, 50S) The first amino acid formylmethionine (fmet) Initiation factors IF1, 2, 3 Selection of Shine-Dalgarno the start codon sequence Assembly of 30S + mRNA + fmet-tRNA initiation complex or 30S + fmet-tRNA + mRNA

Translation properties in prokaryotic cells Elongation factors EF1 (Tu), EF2 (Ts), EF3 (G) Release factors RF1, RF2, RF3, RRF Location in the cell Cytoplasm Transcription and translation are coupled in eukaryotic cells eEF1, eEF2 eRF1, eRF3 Cytoplasm, ER, mitochondria Transcription and translation are spatially and temporally separated. Transcription occurs in the nucleus to produce a pre-mRNA molecule. The pre-mRNA is typically processed to produce the mature mRNA, which exits the nucleus and is translated in the cytoplasm.

Processing of proteins Processing of proteins is a complex of post-translational modifications of the protein molecules (protein maturation)

Steps of processing Proteolytic cleavage (removing segments of the polypeptide chain by enzymes called proteases) Chemical modification (Individual amino acids in polypeptidemight be modified by attachment of new chemical groups) Folding (a process in which a polypeptide folds into a specific, stable, functional, three-dimensional structure; formation of tertiary structure of the protein) by foldases and chaperones Association of protein subunits