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GENE EXPRESSION

Dolores V. Viliran, MD
Department of Biochemistry & Nutrition FEU-NRMF,Institute of Medicine

INTRODUCTION

5’ G~C A~T

3’

3.4 nm

3’

5’

Hydrogen bonds

INTRODUCTION

DNA reverse transcription replication DNA protein

RNA

translation

Central Dogma of Molecular Genetics

SCHEMA OF GENE EXPRESSION

Replication
(DNA Synthesis)

Four Phases Of The Eukaryotic Cell Cycle
 M (mitotic) phase – cell division occur  G1 (gap) phase – longest part of cell cycle
1. Responsible for variation in cell cycle
(8 hrs to >100 days)

2. Cell’s irreversible decision to proliferate is made here
- aided by cell division cycle proteins (CDC) which helps cell to get beyond “restriction point” - CDCs are protein kinases

B. Gap Phase con’t.
3. Cell’s may enter a quiescent phase (Go) – cells do not divide e.g. neurons & muscle cells Factors that bring about quiescence
a) short supply of nutrients b) “contact inhibition” – cell is in contact with other cells

C. S (synthetic) phase – only period when DNA is synthesized
Factors that induce DNA synthesis
2. 3. 4. Carcinogens and tumor viruses Surgical removal of a tumor Mitogens – proteins that bind to cell surface receptors and induce cell division

Types of Replication
1. Conservative replication – one daughter DNA conserves the parental DNA while the other daughter DNA gets the newly synthesized DNA 2. Semiconservative replication – each daughter DNA conserves one strand of parental DNA and one strand of newly synthesized DNA * type of replication in living organisms

Replication fork

REPLICATION
formation of a new DNA strand using another DNA as template occurs at the S phase [cell cycle] location: nucleus, mitochondrion & chloroplast in eukaryotes semi-conservative [Messelson & Stahl] rate: 500 nts are added per second few hrs to duplicate the human genome error rate: 1 mispaired base in 109 nts results to a 4n chromosome number

REPLICATION: Initiation
occurs at the oriC [E coli], ARS [fungi] numerous origins in one chromosome oriC: three 13-bp sequences - first to melt five 9-bp sequences - binding sites for DnaA proteins involved: 1. Topoisomerases - removes the supercoils 2. DnaA - ~ 30 units form a barrel → DNA wounds 3. DnaB - a Helicase; unwinds the DNA & breaks the 4. 5.
hydrogen bonds between the bases DnaC - binds transiently to the DNA SSB - prevents renaturation of the unwound DNA

RNA Primer - ~10 nts synthesized by Primase

REPLICATION: Elongation
5’
LAGGING STRAND

SSB

Ligase
Okazaki fragment

3’ helicase 5’

DNA Polymerase III

DNA Polymerase I primase

Direction of replication fork

3’
LEADING STRAND

REPLICATION: Elongation
DNA Polymerase III polymerase function: 5’ → 3’ [α] proofreading function: 3’ → 5’ exonuclease [ε] Primase - synthesizes the RNA primer DNA Polymerase I - replaces the primer with DNA DNA Ligase - connects adjacent okazaki fragments Eukaryotic DNA Polymerase DNA Pol α - priming DNA Pol β - DNA repair DNA Pol γ - mitochondrial DNA replication DNA Pol δ - main replicative enzyme DNA Pol ε - unknown function

REPLICATION: termination
termination sites [Ter A - E] which are binding sites for TUS [terminator utilization substances] Shortening of the Telomere

Things to remember …...
replication starts at the oriC and ends at the Ter DNA Polymerase III is the main replicative enzyme direction of replication: 5’ → 3’ templates used: both strands reqts for initiation: template, RNA primer, DNA Pol III leading strand - replicated continuously lagging strand - replicated discontinuously [okazaki] linear chromosomes tend to shorten after replication Tx: telomerase - not found in all cells Fidelity: proofreading, excision repair, mutator proteins

Legend

Dna replication in action

Gene Transcription
(RNA Synthesis)

Transcription
• Biosynthesis of RNA • RNA copies of selected DNA sequences (genes) are made

Gene
• Classical definition – A gene is a segment of DNA that determines a single character or phenotype e.g., eye color, facial features, height, etc.

Gene
• Molecular definition – A gene is a segment of DNA that codes for:
• One enzyme • One protein • One polypeptide • One RNA

Types of Gene from Standpoint of Function
A. Structural Genes
– Codes for polypeptides and RNA’s

B. Nonstructural Genes
Operator gene – one that serves as a starting point for reading the genetic code Regulator gene – one that synthesizes the repressor (a substance which switches off the activity of the structural gene).

Definition of Terms
• Cistron
– Smallest unit of DNA which must be intact so that it can serve as transmitter of genetic information

• Muton
– Smallest unit of DNA whose alteration can give rise to a mutant form of organism

• Recon
– Smallest unit of DNA capable of recombination, presumably a series of three nucleotide bases (triplet).

• Recombination
– Formation of a new combination of genes

• Introns
– Intervening segments of DNA that do not code for the amino acid sequence of the polypeptide (function is probably regulatory)

• Exons
– Coding segments of the gene

• Chromosome
– A structure in the nucleus containing the chromatin material made up of: 65% protein (histones) 35% DNA 5% RNA – There are so many genes in a single chromosome, e.g. E. coli = 3,000 to 5,000 genes – Chromatin fibers resemble a string of beads

• Nucleosome
– Repeating beadlike structures composed of: – Segment of DNA duplex (about 200 base pairs) – Eight histone molecules (two each of H2A, H2B, H3 and H4)

• Histones
– Found only in eukaryotes – Function is to package and order DNA into structural units called nucleosomes – H1 = lysine-rich – H2A and H2B = large amount of lysine and arginine – H3 and H4 = arginine-rich

TRANSCRIPTION
formation of RNA using a DNA as template involves a short fragment of the genetic material only only one of the 2 strands is used as template direction of transcription: 5’ → 3’ location: nucleus, mitochondria, chloroplasts occurs in most phases of the cell cycle regulation: via Methylation of the CpG islands of genes housekeeping genes are never methylated rate: ~ 1,500 nts are transcribed in 50 seconds

TRANSCRIPTION
DNA-dependent RNA Polymerase [RNAP] main enzyme involved in transcription components: 1) core enzyme
- ααββ’ - polymerase function 2) sigma factor - regulatory function

no proofreading capacity [less accurate than DNA Polymerase III; prone to more errors] Eukaryotic RNAPs: RNAP I - transcribes rRNAs RNAP II - transcribes mRNAs RNAP III - transcribes tRNAs

A typical transcriptional unit
promoter Informative sequence terminator

DNA

Transcriptional start point

-100
GC

-80
CAAT

-10
TATA

+1

+N

TATA box - aka Pribnow box; recognition site of sigma factor CAAT box - influences rate & frequency of transcription GC box octomers

A typical transcriptional unit
promoter Informative sequence terminator

DNA

GGCCGGCCGGCC------AAAAAAAAAAAAA

GC-rich region = allows hairpin loop formation poly-A tract = allows dissociation of RNAP

A typical transcriptional unit
promoter Informative sequence terminator

DNA

Initiation codon

Polyadenylation signal

Stop codon

Primary transcript

AAAAAAAAA
Mature mRNA

DNA before transcription

A C A T C G A C G C GC A C A 5’ 3’

3’

T

G T A G C T G C G C G T G T

5’

During transcription, The DNA should unwind so that one of its strand can be used as template to synthesize a complementary RNA.
C A T C G A C G

A C A T C G A C G C GC A C A 5’

RNA
5’ C A U C 3’

3’

3’

T

G T A G C T G C G C G T G T

5’

G T A G C T G C

TRANSCRIPTION: steps
δ factor RNAP
CLOSED promoter complex

OPEN promoter complex

Initiation

Elongation

TRANSCRIPTION

TRANSCRIPTION
Termination Signals 1) Rho-independent = GC-rich + poly-A tract 2) Rho-dependent = GC-rich + Rho protein Modification of mRNAs 1) Capping = addition of 5’-methylguanosine cap; occurs after the 5’ end has been synthesized 2) Polyadenylation = addition of the 3’-poly-A tail; requires a clipping enzyme [5’-AAUAAA-3’] and the Poly-A polymerase 3) Splicing of exons = removal of the introns and splicing of the exons; follows the AG-TG rule

TRANSCRIPTION
Splice junctions: “AG-TG rule” Exon 1 GT A Intron subsequent to capping & polyadenylation prior to migration from the nucleus req’ts: small nuclear ribonucleoprotein particles [snRNPs] and snRNAs [U1,U2, U4, U5 and U6] AG Exon 2

COMPARISON
Location Enzyme Direction Template Replication nucleus, mt, chpt DNA Pol III 5’ → 3’ both strands Transcription nucleus, mt, chpt RNAP 5’ → 3’ one strand antisense Primer needed ↑↑↑ dNTPs: thymine not required a gene ↑ NTPs: uracil

Target seq. entire genome Accuracy Units

TRANSLATION
synthesis of a polypeptide based on an mRNA template location: cytoplasm [ribosomes] requirements: 1) mRNA - with codons 2) tRNAs - with anti-codon 3) amino acids main enzyme: peptidyl transferase translational rate: ~ 15 amino acids/second in E coli at 37o C

GENETIC CODE
- maps DNA sequences to proteins in the living celL - It is the sequence of bases (CODONS) in the mRNA which determines the protein that will be synthesized CODON - 3 base words (43) - 64 possible trinucleotide sequences are present 61 = code for amino acid 3 = stop codons Starting codon = AUG - marks the start of a protein sequence End Codons = UAA, UAG, or UGA

Properties of Genetic code
• Degenerate – amino acids are coded by several codon or some codons may code for the same amino acid e.g. UUA,UUG,CUU,CUC,CUA and CUG codes of leucine • • Specific – Each codon signals for a specific amino acid Non-overlapping and without punctuation/commaless – the mRNA coding sequence is “read” by a ribosome starting from the initiating codon (AUG) as a continuous sequence taken three bases at a time until a stop codon is reached. Reading frame – set of continuous triplet codons in a mRNA Universal – Examinations of the translation process in the species that have been investigated have revealed that the coding signals for amino acids are always the same. [though there are some exceptions, especially in the mitochondrial and chloroplast DNA],

• Codons – Anticodon Interaction The base pairing between the anticodon of the tRNA and the mRNA codon is responsible for the actual translation of the genetic information of structural genes. Although codon-anticodon pairings are antiparallel, both sequences are given in the 5’ -> 3’ direction. ex. AUG binds to anticodon CAU - the pairing of the codon (AUG) and anticodon (CAU) ensures that the amio acid will be incorporated into a growing polypeptide chain.
• Most cells possess about 50 tRNAs and some of the anticodon contains uncommon nucleotides such as inosinate (I) which typically occur at the third anticodon.

Codon-anticodon
mRNA
5’ 3’

A=====U tRNA

U=====A

G=====C

5’ 3’

WOBBLE HYPOTHESIS - The first two bases are important in identifying their cognate amino acids The third base [called the wobble base] is not as important, pursuant to the Wobble theory of the genetic code Wobble theory suggested that while the interaction between the codon in the mRNA and the anticodon in the tRNA needed to be exact in two of the three nucleotide positions, this did not have to be so in the third position

-

- The theory proposed that non-standard base-pairing might occur between the nucleotide base in the 5' position of the anticodon and the 3' position of the codon.

5’ anticodon base

3‘ codon base

A

U

C

G

G

C or U

U

A or G

I

A or C or U

TRANSLATION
Messenger RNA [mRNA]
Initiation codon
Stop codon

SD AUG

UGA

PAS

AAAA

codons Shine Dalgarno Sequence - the ribosome binding site AUG - initiation codon; contained within the Kozak sequence in eukaryotes Stop codons - UGA, UAG, UAA PAS - Polyadenylation signal NOTE: mRNA is derived from heterogeneous nuclear RNA [hnRNA] in eukaryotes

TRANSLATION
Transfer RNA [tRNA] cloverleaf appearance adaptor molecule ~ 61 types of tRNAs Pre-Initiation Phase: Charging of tRNA

TRANSLATION
Ribosomes prokaryote small subunit large subunit complete unit 30S 50S 70S eukaryote 40S 60S 80S

the small and large subunits combine after the initiation of translation small subunit = + mRNA binding site [16S RNA] large subunit = + tRNA binding sites [P & A sites] 16S rRNA of the small subunit is highly conserved

Initiation Step
1) Dissociation of 70S [IF1] 2) Binding of IF3 to 30S

3) Binding of IF1 & IF2-GTP

4) Formation of the PreInitiation Complex

5) Binding of 50S to 30S & loss of IF1 & IF3

6) Loss of IF2 and hydrolysis of GTP →GDP + Pi

Elongation
1) Second aa-tRNA enters the A site

2) Peptide bond formation via Peptidyl transferase [23S rRNA of the large subunit]

3) mRNA moves to the left

Termination
occurs when a stop codon occupies the A site
UGA - “opal” codon UAG - “amber” codon UAA - “ochre” codon

no tRNA has the anticodon for the stop codons a release factor recognizes these stop codons, attaches to the A site and breaks up the complex

Release factor

UGA

AAAA

Post-translational Modification
Protein folding - proper folding of nascent polypeptide
made possible by molecular chaperones HSP 70 - prevents premature folding GroeL/GroeS - cage-like structures that prevents aggregation of unfolded proteins

Intein Splicing - removal of inteins and the ligation of exteins
Inteins - intervening, non-informative aa sequences Exteins - informative aa sequences

Signal peptide: ~ 20 aa, for proteins destined for
endomembrane system or for secretion; recognized by the Signal Recognition Peptide [SRP; brings the ribosome to ER]

Proteolytic cleavage - polyproteins are cleaved to yield
several active proteins [ex. Proopiomelanocortin]

ANTIBIOTICS BY SITE OF ACTION
• A. Protein synthesis inhibitors
– 30S ribosomal subunit
• Aminoglycosides(streptomycin), tetracycline

– 50S ribosomal subunit
• Chloramphenicol – binds tp 50S and inhibits peptidyl transferase • Erythromycin and Clindamycin – binds to 50 S and prevent translocation

ANTIBIOTICS BY SITE OF ACTION
• A. Protein synthesis inhibitors
– 60S ribosomal subunit
• Cycloheximide – binds to 60S,inhibits peptidyl transferase

– A and P sites
• Puromycin – an amino acid analog, binds A site,prevents eukaryotic and prokaryotic translation

– eEF-2
• Diphtheria toxin – inhibits eukaryotic elongation factor 2

ANTIBIOTICS BY SITE OF ACTION
• RNA synthesis inhibitors
– Prokaryotic RNA polymerase
• Actinomycin – binds to DNA • Rifampin – binds to β-subunit of RNA polymerase

– Eukaryotic RNA polymerase II
• Amanita phylloides- “angel of Death” mushroom – inhibits RNA pol II

ANTIBIOTICS BY SITE OF ACTION
• DNA synthesis inhibitors
– DNA gyrase inhibitors
• Quinolones

– DNA topoisomerase inhibitors
• Nalidixic acid • Novobiocin

Other Medications: inhibits deoxythymidylate synthesis
• 1. Folic acid analog • inhibitor of dihydrofolate reductase
– Methotrexate – Aminopterin

• 2. Inhibits Thymidylate Synthesis
– 5-Fluorouracil

LIST OF INHIBITORS
Coumarine Quinolones Rifampicin inhibits DNA gyrase by competing with ATP inhibits DNA gyrase by binding its amino terminus inihibits RNA polymerase by binding to its β chain

Chloramphenicol binds to 50S and prevents peptide bond formation Erythromycin inhibits translocation of 50S subunit Fusidic acid inhibits translocation by preventing dissociation of EF-G-GDP from ribosome Puromycin an aa-tRNA analog causing premature chain term’n Tetracycline binds to 30S subunit and prevents aa-tRNA binding to the A site Cyclohexamide inhibits peptidyl transferase on eukaryotic 60S Diphtheria toxin inhibits eukaryotic EF-2 by ADP ribosylation Streptomycin binds to 30S subunit & causes mRNA misreading Kanamycin binds to 70S unit and causes mRNA misreading

mRNA

Ribosome binding protein

AUGUCUCCGUAA

Small ribosomal subunit Large ribosomal subunit

AUGUCUCCGUAA
Small ribosomal Subunit binds to mRNA

Large ribosomal subunits binds to small subunit forming complete ribosomes

AUGUCUCCGUAA

P – site on ribosomes

A – site on ribosomes

AUGUCUCCGUAA

UAC
Anti-codon

codons

tRNA

Amino Acid (Met)

First, tRNA binds at the P-site

AUGUCUCCGUAA

UAC

Anti-codon matches initiating codon on mRNA

AUGUCUCCGUAA UAC

AGA

Next tRNA with different anti-codon and amino acid

AUGUCUCCGUAA UAC
Second tRNA binds in the A – site of the ribosome and matches the next codon

AGA

AUGUCUCCGUAA U A CA G A

Amino acid moved from first tRNA and joined to second tRNA forming a di-peptide

AUGUCUCCGUAA U A CA G A

Ribosomes moves and the tRNA with dipeptide moves to P-site

Empty tRNA is released

AUGUCUCCGUAA AGA GGC

Third tRNA with amino acid arrives

AUGUCUCCGUAA AGA

Codon matches anti-codon

GGC

tRNA moves onto A-site

Third tRNA with amino acid arrives

AUGUCUCCGUAA AGA GGC

Di peptide moved and Tri-peptide formed

AUGUCUCCGUAA AGA GGC
Ribosomes moves

tRNA moves to P-site

empty tRNA moves off ribosome

AUGUCUCCGUAA GGC AGA
Ribosomes moves Termination signal no anti-codon on any tRNA

tRNA moves to P-site

empty tRNA moves off ribosome

AUGUCUCCGUAA GGC

Complete polypeptide is released

AUGUCUCCGUAA GGC

Ribosome units separate

Complete polypeptide is released

What is the possible role of modified histones?
A. methylation of histones is correlated with activation and repression of gene transcription B. Methylation of histones leads to transcription activation only C. Phosphorylation of histones H3 and H4 is associated with the condensation of chromosomes during the replication cycle D. Acetylation of histone H1 is associated with chromosomal assembly during DNA

What is the possible role of modified histones?
A. methylation of histones is correlated with activation and repression of gene transcription B. Methylation of histones leads to transcription activation only C. Phosphorylation of histones H3 and H4 is associated with the condensation of chromosomes during the replication cycle D. Acetylation of histone H1 is associated with chromosomal assembly during DNA

Which is considered as transcriptionally active chromatin
A. euchromatin B. heterochromatin C. constitutive chromatin D. telomeres

Which is considered as transcriptionally active chromatin
A. euchromatin B. heterochromatin C. constitutive chromatin D. telomeres

The genetic code is characterized as:
A. non-degenerate B. non-overlapping C. species-specific D. non-specific

The genetic code is characterized as:
A. non-degenerate B. non-overlapping C. species-specific D. non-specific

The precise initiation codon is determined by:
A. shine-Dalgarno sequence B. Kozak sequence C. Consensus sequence D. Repetitive sequence

The precise initiation codon is determined by:
A. shine-Dalgarno sequence B. Kozak sequence C. Consensus sequence D. Repetitive sequence

The first tRNA that enters the P site during translation process.
A. Lys tRNA B. Met tRNA C. Ala tRNA D. Glut RNA

The first tRNA that enters the P site during translation process.
A. Lys tRNA B. Met tRNA C. Ala tRNA D. Glut RNA

Factors that can cause DNA denaturation:
A. Alkaline pH B. High temperature C. Acidic pH D. A and B E. B and C

Factors that can cause DNA denaturation:
A. Alkaline pH B. High temperature C. Acidic pH D. A and B E. B and C

Which enzyme is needed for synthesis of the eukaryotic DNA lagging strand
A. DNA polymerase alpha B. DNA polymerase sigma C. DNA polymerase III D. DNA polymerase

Which enzyme is needed for synthesis of the eukaryotic DNA lagging strand
A. DNA polymerase alpha B. DNA polymerase sigma C. DNA polymerase III D. DNA polymerase

Which enzyme can remove the supercoiling produced during replication
A. Helicase B. Topoisomerase C. Primase D. Single-strand binding protein

Which enzyme can remove the supercoiling produced during replication
A. Helicase B. Topoisomerase C. Primase D. Single-strand binding protein

Matching Type: __16. mRNA serves as template __17. TATA Box plays a crucial role __18. May or may not require a rho facto __19. This is initiated with activation step __20. An essential process in cell divisio

A. Transcription B. Translation C. Replication D. A/B E. A/B/C

Matching Type: _B_16. mRNA serves as template A. Transcription _A_17. TATA Box plays a crucial role Translation _A_18. May or may not require a rho facto Replication _B_19. This is initiated with activation step A/B _C_20. An essential process prior to division of cell A/B/C

B. C. D. E.

Any Questions ?