MOLECULAR GENETIC PART I

Gene Regulation in Protein Synthesis

1. Gene Regulation in Protein Synthesis

A. Nucleic Acid Structure B. Replication of DNA C. Transcription of Bacterial DNA D. Transcription of Eukaryote DNA E. Translation

A. Nucleic Acid Structure

Two types of nucleic acids- DNA & RNA DNA (DeoxyriboNucleic Acid) & RNA (RiboNucleic Acid) Covalent link between atoms IN nucleic acids building blocks protect the biological polymers Weak bond BETWEEN different parts allow functions

DNA
1. Molecule in which the genetic material is stored 2. Nucleoside: purine base (Adenine, Guanine) pyrimidine base (Thymine, Cytosine) 3. F(x) as a template for its self-replication 4. F(x) as a template for RNA synthesis 5. DNA can adopt different conformations A form, B form, Z form

DNA
6. DNA can exist in two conditions supercoiled & relaxed 7. Naturally occurred in negatively supercoiled condition 8. Enzyme alter DNA structure T opoisomerase I (A & B) for single strand break T opoisomerase II for double strand break, multimeric enzyme, require ATP hydrolysis to complete two DNA strands cleavage Prokaryotes has a special topoisomerase II known as DNA gyrase which introduce negative supercoil

DNA
9. Stabilize by hydrogen bonding, stacking interactions and ionic interactions

10.Undergo denaturation when heated and anneal when cooled

Phosphodiester bond

Hydrogen bond

RNA
Chemically very similar to DNA Three differences: 1. deoxyribose vs ribose backbone 2. thymine vs uracil 3. Typically found in cell as single polynucleotide chain

RNA
Five functions: 1. Intermediate between the gene and the protein synthesizing machinery (mRNA) 2. Adaptor between the codons in mRNA and amino acids (tRNA) 3. Structural role as a components of the ribosomes (rRNA) 4. Regulatory molecule which complement and interferes with the translation of certain mRNAs 5. Enzymes that catalyze essential reactions in the cell (ribozymes)

RNA

F(x) as a template for protein synthesis Comes in various shapes, sizes and some with catalytic property

B. Replication of DNA
THE CHEMISTRY OF DNA SYNTHESIS Requires TWO key substrates dNTP (dATP , dGTP , dCTP , dTTP) and primer:template junction

B. Replication of DNA
THE CHEMISTRY OF DNA SYNTHESIS DNA is synthesized by extending the 3 end of the primer in antiparallel orientation - Matching dNTP form a base pair by hydrogen bonding - hydroxyl group from 3 end of primer attack the P (-phosphoryl) from dNTP to form phosphodiester bond and release the pyrophosphate (- and -phosphoryl)

B. Replication of DNA
THE CHEMISTRY OF DNA SYNTHESIS Hydrolysis of pyrophosphate as a driving force for DNA synthesis - Polymerization of nucleotides is promoted by additional free energy provided by the rapid hydrolysis of pyrophosphate

B. Replication of DNA
INITIATION OF DNA REPLICATION Involves a replicator and initiator components Replicator is a set of cis-acting DNA sequence sufficient to direct an initiation of replication - Binding site for the initiator protein which nucleates the assembly of replication initiation machinery - Consist of AT-rich region which unwind readily

B. Replication of DNA
Initiator is a sequence-specific DNA binding protein - Binds to a specific sequence within the replicator - Unwind the DNA region - Attract other factors required for initiation of replication

B. Replication of DNA
Unwinding of duplex DNA strands catalyzed by DNA helicase T o stabilize the unwound single-strand DNA , a single-strand DNA binding protein (SSBs) bind to ssDNA Replication fork formation Primase synthesis RNA primers on ssDNA to initiate DNA synthesis by DNA polymerase

B. Replication of DNA REPLICATION FORK

DNA is synthesized semidiscontinuously Both DNA strands are synthesized simultaneously The junction between newly separated strands and the unreplicated duplex DNA is known as replication fork

B. Replication of DNA REPLICATION FORK

Synthesis of DNA occur only from 5 to 3 direction. In lagging strand, DNA is synthesized discontinuously and form a short new fragments, known as Okazaki fragments

B. Replication of DNA DNA Polymerase

Key enzyme for DNA replication Have single active site which catalyze the addition of any four dNTPs Bound to primer:template junction on DNA strand Monitor the accuracy of base-pairing by catalysis rate. High rate=matched, slow rate=mismatched

B. Replication of DNA DNA Polymerase

When mismatched occur, primer:template junction will slide out from catalytic site of DNA polymerase into proofreading exonuclease site Exonuclease will remove the unmatched base from 3-end Primer:template junction will slide in into the catalytic site of DNA polymerase

B. Replication of DNA
RNA primers are removed by RNAse H through degradation The final ribonucleotide bond to DNA end is removed by exonuclease which result in gaps DNA ligase use ATP to create phosphodiester bond between 5 phosphate and 3 OH in a newly synthesized DNA strands to close this gap

REPLICATION

TRANSCRIPTION

Synthesis of new Synthesis of new deoxyribonucleotide strand ribonucleotide strand Catalyze by DNA polymerase Catalyze by RNA polymerase DNA polymerase require a RNA polymerase doesnt primer to initiate synthesis require any primer to initiate synthesis The newly synthesized DNA The newly synthesized RNA form a base-pair with DNA doesnt remain base-paired template to the DNA template Involve extensive Lack of extensive proofreading proofreading Copy the entire genome Copy certain parts of the once in every cell division genome

C. Transcription of DNA
Key enzyme is RNA polymerase Involve three phases: 1. Initiation 2. Elongation 3. T ermination

C. Transcription of Bacterial DNA

INITIATION
Binding of polymerase to a promoter sequence, form a closed complex Upon binding, DNA strands separate around the start site and form a transcription bubble (open complex) First two ribonucleotides are bought into active site, aligned and joined Polymerase move ahead opening the DNA duplex

C. Transcription of Bacterial DNA

INITIATION
Formation of first 10 bp ribonucleotides is a rather inefficient process, usually being released and transcription start againabortive initiation When transcribed sequence reach more than 10 bp, a stable ternary complex is established Followed by elongation phase

C. Transcription of Bacterial DNA

ELONGATION
DNA duplex enter polymerase and separated at the catalytic cleft Ribonucleotides enter the site and joined the growing RNA chain guided by the DNA template Only 8-9 growing RNA chain remain on the DNA template and the previously generated are peeled off and directed out from polymerase

C. Transcription of Bacterial DNA

ELONGATION
During elongation, polymerase carries out two proofreading functions: 1. Pyrophosphorolytic editing Polymerase uses its active site to catalyze the removal of incorrect ribonucleotide by incorporating the pyrophosphate (PPi) 2. Hydrolytic editing Polymerase is backtrack by one or more mismatched nucleotides and remove the errorcontaining sequence

C. Transcription of Bacterial DNA

TERMINATION
ermination is triggered by a specific sequence T known as terminators Bacteria: rho-independent OR rho-dependent - Rho-independent involve a stem-loop structure formed by self base-pairing - Rho-dependent require Rho protein, a transcription termination factor which require ATP to remove RNA chain from template and polymerase

D. Transcription of Eukaryote DNA

INITIATION
Polymerase II + general transcription factors + promoter pre-initiation complex Promoter consist of 4 elements: recognition element (BRE), TATA box, initiator (Inr), downstream promoter element (DPE) Formation of pre-initiation complex promoter melting DNA unwinding (require hydrolysis of ATP)

D. Transcription of Eukaryote DNA

INITIATION
In vivo environment, mediator complex and transcriptionally regulatory proteins are needed Different circumstances and promoters requires nucleosome modifiers and chromatin remodellers

D. Transcription of Eukaryote DNA

ELONGATION
Pol II C-terminal domain (CTD) phosphorylated at Ser residue exchange of initiation factors with elongation and RNA processing factors Protein kinase p-TEFb phosphorylate CTD and two other elongation factors stimulate elongation TFIIS proofread RNA sequence by inherence RNAse activity, similar to hydrolytic editing in DNA replication

D. Transcription of Eukaryote DNA

POST ELONGATION
Elongation, termination and RNA processing are interconnected events RNA processing: - 5 capping (+ methylated G to 5 end) - RNA splicing (removal of intron) - 3 polyadenylation (+ A-tail to 3 end) ermination depend on specific sequence T

D. Transcription of Eukaryote DNA

POST ELONGATION
mRNA packaged and exported from nucleus to cytoplasm Require active process (export protein + GTPase) Through a nuclear pore complex

E. Translation of DNA
Generation of amino acid sequences from mRNA Involve mRNA, tRNA, aminoacyl tRNA synthetase and ribosome Translation start at 5 ORF (open reading frame), end at 3 ORF . Start codon: AUG, GUG, UUG Stop codon: UAG, UAA, UGA

E. Translation of DNA
Codons are degenerate. 4 nucleotide with 64 possibilities only code for 20 amino acids.
Peptidyl-tRNA binding site Aminoacyl-tRNA binding site

Exit

E. Translation of DNA

E. Translation of DNA BACTERIA

Translation occur once mRNA emerge from RNA polymerase Ribosome binding to RBS (Shine-Dalgarno sequence 5 AGGAGG 3) Complementary sequence (5 CCUCCU 3) in 16S rRNA bind to this element Initiator tRNA (fMet-tRNA) bind to P site in small ribosome pairing of anticodon with start codon Association of large rRNA subunit GO

E. Translation of DNA EUKARYOTE

Ribosome binding is recruited by 5 cap Translation efficiency increased by Kozak sequence (5 G/ANNAUGG 3) Initiator tRNA (Met-tRNA) bind to P site in small ribosome Recognition mediator proteins bind to mRNA at 5 cap, attract RNA helicase and unwind hairpin structure binding to small ribosome Start scanning until 5 AUG 3 is found

E. Translation of DNA EUKARYOTE

Correct base-pairing of Met-tRNA and 5 AUG 3 result in release of recognition mediator factors Association of large rRNA GO

E. Translation of DNA
ELONGATION and TRANSLOCATION

E. Translation of DNA
TERMINATION
Release factor (RF) recognize stop codon Two class RF: - Class I: recognize stop codon and trigger hydrolysis of peptide chain - Class II: stimulate the dissociation of class I factor from ribosome

DNA MUTATION
Missense mutation: amino acid substitution -eg.: sickle cell anemia (glutamate to valine) Stop mutation: incomplete polypeptide Frameshift mutation: insertion or deletion of one or more base

END OF PART I