Gene Expression PDF

# Gene Expression ## 17- GENE EXPRESSION Synthesis of protein under the influence of gene is called gene expression. It includes RNA transcription and protein synthesis (translation). * **Gene:** is a DNA segment that contained a well defined genetic information & codes for an individual functio...

# Gene Expression ## 17- GENE EXPRESSION Synthesis of protein under the influence of gene is called gene expression. It includes RNA transcription and protein synthesis (translation). * **Gene:** is a DNA segment that contained a well defined genetic information & codes for an individual function e.g. polypeptide chain. * **Genome:** means total DNA content of a cell (= total number of genes in one cell). ## 17 - A-Transcription Transcription is the process of synthesis of RNA from a DNA template by RNA polymerase enzymes and a number of associated proteins. * For any particular gene, only one strand of the DNA molecule, called the template strand, is copied by RNA polymerase. The other strand is referring as coding (antitemplate strand) of that gene. The coding strand is not used during transcription. It corresponds exactly to the sequence of the primary RNA transcript, except that RNA contains uracil instead of the thymine found in DNA coding strand. * DNA - dependent RNA polymerase elongates an RNA strand by adding ribonucleotide units to the strand 3'-hydroxyl end and builds RNA in the 5'3' direction. **Figure. 68: Biosynthesis of RNA (Transcription)** A diagram showing a DNA molecule with the coding strand being transcribed by RNA polymerase. The diagram shows the primary RNA being synthesized. It also explicitly shows that the 3` end of the primary RNA extends beyond the coding strand. **Steps of RNA synthesis:** The general steps required to synthesize the primary transcript (i.e. a newly synthesized RNA) are initiation, elongation and termination. - Initiation protein factors and RNA polymerase are needed for initiation process. ## RNA Polymerase RNA polymerase recognizes and binds to the promoter region (The promoter is the binding site for RNA polymerase on the double - stranded DNA. They have conserved or consensus sequence). **Figure. 69: Transcription of DNA** A diagram showing a DNA molecule with a highlighted promoter region, a terminator, and a coding sequence for an RNA transcript. The 5' and 3’ directions of the DNA template, the coding strand, and the RNA are shown. RNA polymerase is shown transcribing the DNA template. **N.B.:** - The promoter identifies the start site for transcription and orients the enzyme on the template strand. - RNA polymerase then separates the two strands of DNA as it reads the base sequence of the template strand. - Transcription begins at the 1+ base pair. (initiation protein factors are released as soon as transcription is initiated). - The first nucleotide (always purine) associates with the initiation site. Except for the first nucleoside triphosphate, subsequent nucleotides are added to the 3'-hydroxyl of the preceding nucleotide forming phosphodiester bond and pyrophosphates are released. **Elongation:** RNA polymerase continues moving along the template strand in the 5`3` direction, synthesizing RNA in 3' - 5`direction (elongation protein factors are needed in eukaryotes) **Termination:** **Post - transcriptional modification of RNA (RNA processing ):-** The immediate product of transcription is a precursor RNA molecule; the primary transcript, which is modified subsequently to a mature functional molecule. The primary transcript is copied from a linear segment of DNA, a transcription unit, between specific initiation and termination sites. **1- Processing of eukaryotic mRNA precursor (heterogenous nuclear RNA; hn RNA):-** **(1) Capping:-** - The cap (-7methyl guanosine) is added to the 5` end while the RNA molecule is still being synthesized. - 7-methyl guanosine is joined to the 5' end of mRNA in an unusual 5',5'-triphosphate linkage. - All mRNA are capped to be functional in protein biosynthesis. **Significance of the cap structure:-** - Serves as a ribosome binding site (for translation initiation). - Protects the 5' end of mRNA from attack by 5' 3' exonuclease (for mRNA stability). **(2) Polyadenylation:-** Poly (A) polymerase enzyme adds poly (A) tail to the 3'end of mRNA (which is short at first about 20A residues, then subsequently extended to as much as 200 A residues) **Significance of the poly (A) tail:-** - Protects the 3` end of mRNA from 5'- 3' exonuclease attack (for mRNA stability). - Aids in its transport from nucleus to cytoplasm. **Figure. 70: The 5` cap of mRNA** - A visual representation in text form of the 5` cap of an mRNA molecule. The structure of the cap is shown and there are descriptions of the different parts indicated using boldface. **(3) Splicing:** - The coding portions; exons (the sequence of a gene that is represented as mRNA) of most eukaryotic genes are interrupted by intervening sequences; introns (the sequence of a gene that is transcribed but excised before translation). - Splicing means removal of introns from the primary transcript in the nucleus and then ligation of exons to form mature functional m RNA. The mature mRNA is then transported to the cytoplasm where it is translated into protein. **II- Processing of eukaryotic transfer RNA precursor (pre- tRNA):-** Eukaryotic tRNA genes are all transcribed by RNA polymerase III. The primary transcript (pre- tRNA) requires post- transcriptional processing to be mature functional tRNA such as:- - Folding and base pairing to generate its characteristic shape. - Removal of excess nucleotides from the 5` and 3` ends (cleavage). - Removal of introns (splicing). - Addition of the CCA sequence at 3` end. - Modification of some bases by methylation, deamination or reduction. **III- Processing of eukaryotic ribosomal RNA precursor (pre-r RNA):-** - Pre-rRNA (455) - The ribosomal subunits assemble in the nucleolus as rRNA pieces combine with ribosomal proteins. Eukaryotic ribosomal subunits are 60 S and 40 S. They join during protein synthesis to form the whole 80 S ribosome. ## 17 - B- Protein Biosynthesis **Flow of genetic information from DNA to protein:** - DNA molecule (template): Gene 1, Gene 2, Gene 3 - TRANSCRIPTION: A diagram representing the molecule of DNA with the template strand being transcribed. - mRNA: Shows a representation of the mRNA sequence, with codons highlighted. - TRANSLATION: Shows a polypeptide chain with amino acids denoted by their three-letter abbreviations: Trp, Phe, Gly, Ser. # The Genetic Code The genetic information of the cell is stored and transmitted in the nucleotide sequences of DNA. Expression of this genetic informations involves two stages:- - The first stage is transcription to form mRNA that carries specific and precise messages in the form of codons from DNA to the cytoplasmic sites of protein synthesis. - The second stage is translation of the nucleotide sequence of a mRNA (Codons) into an amino acid sequence of a protein. Each codon consists of a sequence of 3 nucleotides i.e. it is a triplet code. Collection of these codons makes up the genetic code. **Note:Thus, protein biosynthesis is also called translation because it involves translation of information from the 4 - letter language and structure of nucleic acid into the -20letter language and structure of proteins** # Components of Translational Process:- - m RNA as a carrier of genetic information. - t RNA as an adapter molecule, which recognizes an amino acid on one end and its corresponding codon on the other end. - Ribosomes as the molecular machine coordinating the interaction between mRNA, tRNA, the enzymes and the protein factors required for protein synthesis. **Genetic Code** It is the relation between sequence of nucleotides in DNA (or in m RNA) and the sequence of amino acids in a polypeptide chain. (table below). **First Nucleotide** - **Second Nucleotide** - **Third Nucleotide** **U** - **U:** Phe, Ser, Tyr, Cys - **C:** Phe, Ser, Tyr, Cys - **A:** Leu, Ser, Term, Term2 - **G:** Leu, Ser, Term, Trp **C** - **U:** Leu, Pro, His, Arg - **C:** Leu, Pro, His, Arg - **A:** Leu, Pro, Gln, Arg - **G:** Leu, Pro, Gln, Arg **A** - **U:** Ile, Thr, Asn, Ser - **C:** Ile, Thr, Asn, Ser - **A:** Ile2, Thr, Lys, Arg2 - **G:** Met, Thr, Lys, Arg2 **G** - **U:** Val, Ala, Asp, Gly - **C:** Val, Ala, Asp, Gly - **A:** Val, Ala, Glu, Gly - **G:** Val, Ala, Glu, Gly **The terms first, second, and third nucleotide refer to the individual nucleotides of a triplet codon. U, uridine nucleotide; C, cytosine nucleotide; A, adenine nucleotide; G, guanine nucleotide; Met, chain initiator codon; Term, chain terminator codon. AUG, which codes for Met, serves as the initiator codon in mammalian cells.** # Protein Biosynthesis It can be described in 3 phases; initiation, elongation and termination. The protein sequence is synthesized and read from the amino terminus to the carboxy terminus. **I. Initiation:** For initiation of protein biosynthesis, there must be:- - tRNA, rRNA, mRNA, Eukaryotic initiation factors (elFs), factors, every RNA, energy, diff AA, ammocupletes - GTP, ATP and different amino acids. • tRNA charging It means recognition and attachment of the specific amino acid to the 3 hydroxyl adenosine terminus ( to the sugar) of tRNA in an ester linkage.) **Amino acid (AA)** **Initiation involves 4 steps:-** 1. **Ribosomal dissociation:** The 80 S eukaryotic ribosome is dissociated into 40 S and 60 S subunits. elF –3 and elF1- bind to 40 S subunit thus preventing reassociation between the 2 subunits 2. **Formation of 43 S pre initiation complex:** The first step in this process involves the binding of GTP by elF2-. This binary complex then binds to Met – tRNA (a tRNA specifically involved in binding to the initiation codon AUG). This ternary complex binds to the 40 S ribosomal subunit to form preinitiation complex. 3. **Formation of 48 S initiation complex:** mRNA binds to the preinitiation complex to form the 48 S initiation complex with hydrolysis of ATP to ADP + Pi. This initiation complex scans the mRNA for a suitable initiation codon. 4. **Formation of 80 S initiation complex:** The binding of 60 S ribosomal subunit to the 48 S initiation complex involves the hydrolysis of the GTP bound to elF2-. This reaction results in the release of the initiation factors bound to 48 S initiation complex (these factors are recycled) and rapid association of 40 S and 60 S subunits to form the 80 S ribosome. At this point, Met - tRNA is on the peptidyl (P) site of ribosome, ready for the elongation cycle to commence. **II. Elongation:** - It is a cyclic process involving 3 steps 1. **Binding of aminoacyl - tRNA to the A site:** In the complete 80 S ribosome formed during initiation, the aminoacyl (A) site is free. The binding of the proper aminoacyl tRNA in the A site requires:- • Proper codon recognition. • Activation of aminoacyl tRNA by binding of eukaryote elongation factor-1 (e EF1-) and GTP (ternary complex). When aminoacyl tRNA binds to A site, GTP is hydrolysed and e EF1- is released. **2. Peptide bond formation:** - The a-amino group of the new aminoacyl tRNA in the A site attacks the carboxyl group of the peptidyl- tRNA occupying the P site. This reaction is catalyzed by peptidyl transferase of the 60 S subunit (this is an example of ribozyme activity) The reaction results in attachment of the growing peptide chain to the tRNA in the A site. **3- Translocation:** - Upon removal of the peptidyl moiety from the tRNA in the P site, the discharged tRNA quickly dissociates from the P site. eEF2- and GTP are responsible for the translocation of the newly formed peptidyl tRNA at the A site into the empty P site. The translocation of the newly formed peptidyl tRNA and its corresponding codon into the P site then frees the A site for another cycle of amino acyl - tRNA codon recognition and elongation. - The formation of one peptide bond requires energy resulting from hydrolysis of 4 high energy phosphate bonds:- - Charging of tRNA with amino acyl moiety requires hydrolysis of an ATP to an AMP. - The entery of amino tRNA into the A site requires one GTP hydrolysis to GDP. - The translocation of the newly formed peptidyl - tRNA in the A site into the P site results in hydrolysis of one GTP to GDP. **Figure. 72: Diagrammatic representation of the peptide elongation process of protein synthesis the small labeled n1-,n,n1+, etc represent the amino acid residues of the newly formed protein molecule EF1- and EF2- represent elongation factors 1 and 2, respectively. The peptidyl-tRNA and aminoacyl-tRNA sites on the ribosome are represented by P site and A site, respectively** - A diagram showing a ribosome with tRNA molecules in site P and site A. The diagram also shows the involvement of elongation factors 1 and 2 in the translocation process. **III. Termination** - After many cycles of elongation, the non-sense or termínation codon of mRNA (UAA, UAG or UGA) appears in the A site. Normally, there is no tRNA with an anticodon capable of recognizing such a termination signal. Releasing factors (eRFS) can recognize the termination signals in the A site. eRF, GTP and peptidyl transferase promote the hydrolysis of the bond between the peptide and the tRNA occupying the P site. Then the 80 S ribosome dissociates into 40 S and 60 S subunits and mRNA, tRNA, eRF, GDP and Pi are released. **Site of protein biosynthesis:-** - Protein biosynthesis takes place on ribosomes. Many ribosomes can assemble to translate a single mRNA molecule forming a polysome. Polysomes are either found free in cytoplasm or attached to rough endoplasmic reticulum, depending on the protein being translated. The free polysomes are responsible for the synthesis of proteins required for intracellular functions, while attached polysomes synthesize integral membrane proteins and proteins to be exported. **Protein maturation:-** - Newly synthesized polypeptide chains undergo folding and posttranslational processing. These modifications may result in their activation to functional form, localization in subcellular compartment or their secretion from the cell. **Figure. 73: Termination of Protein Synthesis** - A diagrammatic representation of the termination of protein synthesis. It shows an incoming releasing factor interacting with a ribosome containing a tRNA in the P site and a termination codon in the A site and a polypeptide chain being released from the ribosome. **I- Protein folding:** - As proteins emerge from ribosomes, they fold into three-dimensional conformations that are essential for their subsequent biologic activity. - Molecular chaperones are proteins which assist in the process of proper protein folding. - Misfolded proteins are targeted for destruction. **II- Post - translational processing:** **(1) Proteolysis:** proteolysis of proteins is a common maturation step. - Amino terminal, carboxy terminal or internal sequences can be removed from the protein. **Examples:** 1. Removal of amino terminal Met residues of most eukaryotic proteins. 2. Removal of signal peptides by signal peptidases. - (Signal sequence which directs the protein to its appropriate location in the cell is often removed by signal peptidase after the protein has reached its final site). **(2) Modifications of individual amino acids:-** **Examples:** - Hydroxylation of proline and lysine to form hydroxyproline and hydroxylysine is important for collagen synthesis. - Phosphorylation of serine, threonine or tyrosine by ATP – requiring protein kinases regulates the activity of many enzymes e.g. glycogen phosphorylase. - y-Carboxylation of glutamic acid residues in prothrombin to form y- carboxy glutamate; allows binding of calcium ions and leads to formation of blood clot. **(3) Addition of certain groups:** - Attachment of carbohydrate side chains (Glycosylation) to proteins forms glycoproteins. In some glycoproteins, the carbohydrate side chain is attached to asparagine residues (N-linked), in others to serine and threonine residues (O-linked). **• Inhibitors of protein biosynthesis:** - Effective antibiotics must interact specifically with prokaryotic ribosomes to inhibit bacterial protein synthesis without interaction with components of eukaryotic ribosomes (e.g. streptomycin, Tetracyclin, chloramphenicol and erythromycin). - Streptomycin inhibits initiation of prokaryotic protein biosynthesis. - tetracyclines block the A site on prokaryotic ribosome, thus inhibit the binding of amino acyl t RNA. - Chloramphenicol blocks peptidyl transfer on prokaryotic ribosomes. - Erythromycin inhibits the translocation reaction of prokaryotic protein synthesis. - Other antibiotics are not clinically useful because they inhibit protein synthesis in both prokaryotes as eukaryotes (as puromycin) or only in eukaryotes (as cycloheximide). - Some toxins inhibit eukaryotic protein synthesis (as diphtheria toxins and ricin).