CELL CYCLE AND DOGMA BIOLOGY. Cell Cycle dividingnon-dividingThe dividing and non-dividing stages in...

CELL CYCLE AND CELL CYCLE AND DOGMA BIOLOGYDOGMA BIOLOGY

Cell CycleCell Cycle

• The dividingdividing and non-dividingnon-dividing stages in the life of a cell.

• Phases:

1.1. InterphaseInterphase: growth and DNA replication

2.2. ProphaseProphase

3.3. MetaphaseMetaphase

4.4. AnaphaseAnaphase

5.5. TelophaseTelophase

Mitotic divisionMitotic division

Eukaryotic Cell Cycle

1. Cell growth2. Chromosome replication3. Cell division (2 types)

• Mitosis• Meiosis

Cell CycleCell Cycle

Mitosis-prophase-metaphase-anaphase-telophase

G1 G2

Sphase

interphase

Cell Cycle Phases

• Interphase: cell growth & DNA replication

• Mitosis : nuclear & cell Division

InterphaseInterphase

• Comprises about 90%90% of the cell cyclecell cycle.• Cellular growthCellular growth:

a. protein synthesisprotein synthesis

b. metabolic activitiesmetabolic activities

c. DNADNA synthesissynthesis• Made up of three phasesthree phases:

1. G1. G11 phase phase

2. S phase2. S phase

3. G3. G22 phase phase

InterphaseInterphase

1. G1. G11 (gap) phase: (gap) phase:

a. protein synthesis and metabolic activities.

b. most cell are arrested in this phasemost cell are arrested in this phase.

2. S phase:2. S phase:

DNA DNA replication replication takes place.

3. G3. G22 (gap) phase: (gap) phase:

Cellular growth and preparation for M phaseM phase.

The Central Dogma in Biology

ProteinRNATranscription Translation

DNA

Replication

Preserving information is expensive:Formation of any peptide bond 21 KJ/molFormation of a peptide bond b/n 2 specific amino acids 125 KJ/mol

Central Dogma (Modifications)

Transcription TranslationDNA

(1) Reverse transcription

Replication

RNA

(2)Self Replication

Protein

(3)Self Replication

(2)Ribozymes

DNA DNA ReplicationReplication

DNA Replication

• Origins of replicationOrigins of replication

1. Replication ForksReplication Forks:

hundredshundreds of Y-shapedY-shaped regions of

replicating DNA moleculesreplicating DNA molecules where new

strands are growing.

ReplicationReplicationForkFork

Parental DNA MoleculeParental DNA Molecule

3’

5’

3’

5’

http://www.ultranet.com/~jkimball/BiologyPages/D/DNAReplication.html

DNA Replication

• Origins of replicationOrigins of replication

2. Replication BubblesReplication Bubbles:

a.HundredsHundreds of replicating bubbles

(Eukaryotes)(Eukaryotes).

b.SingleSingle replication fork (bacteria).(bacteria).

Bubbles Bubbles

http://faculty-staff.ou.edu/S/Barbara.Safiejko-Mroczk-1/3113/Lectures/Lecture%2013/13.dna.replicat.html

DNA ReplicationDNA Replication

• Strand SeparationStrand Separation:

1.1. HelicaseHelicase:

enzyme which catalyze the unwindingunwinding and

separationseparation (breaking H-Bonds) of the parental

double helix.

2.2. Single-Strand Binding ProteinsSingle-Strand Binding Proteins:

proteins which attach and help keep the

separated strands apart.


• Strand SeparationStrand Separation:

3.3. TopoisomeraseTopoisomerase: enzyme which relieves relieves stressstress on the DNA moleculeDNA molecule by allowing free rotation around a single strand.

Enzyme

DNA

Enzyme


• Priming:Priming:

1.1. RNA primersRNA primers: before new DNA strands can form, there must be small pre-existing

primers (RNA)primers (RNA) present to start the addition of new nucleotides (DNA Polymerase)(DNA Polymerase).

2.2. PrimasePrimase: enzyme that polymerizes (synthesizes) the RNA Primer.


• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:

1.1. DNA PolymeraseDNA Polymerase: with a RNA primerRNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the synthesis of a new DNA strand in the

5’ 5’ to 3’ directionto 3’ direction.

RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase

NucleotideNucleotide

5’

5’ 3’

NucleotideNucleotide

OO=P-O O

PhosphatePhosphate GroupGroup

NNitrogenous baseNitrogenous base (A, G, C, or T)(A, G, C, or T)

CH2

O

C1C4

C3 C2

5

SugarSugar(deoxyribose)(deoxyribose)

DNADNA

P

P

P

O

O

O

1

23

4

5

5

3

3

5

P

P

PO

O

O

1

2 3

4

5

5

3

5

3

G C

T A



2.2. Leading StrandLeading Strand: synthesized as a single polymersingle polymer in the 5’ to 3’ 5’ to 3’

directiondirection.

RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides

3’5’

5’



3.3. Lagging StrandLagging Strand: also synthesized in the 5’ to 3’ direction5’ to 3’ direction, but

discontinuouslydiscontinuously against overall direction of replication.

RNA PrimerRNA Primer

Leading StrandLeading Strand

DNA PolymeraseDNA Polymerase

5’

5’

3’

3’

Lagging StrandLagging Strand

5’

5’

3’

3’



4.4. Okazaki FragmentsOkazaki Fragments: series of short segments on the lagging strand.lagging strand.

Lagging Strand

RNARNAPrimerPrimer

DNADNAPolymerasePolymerase

3’

3’

5’

5’

Okazaki FragmentOkazaki Fragment

DNA ReplicationDNA Replication• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:

5.5. DNA ligaseDNA ligase: a linking enzyme that catalyzes the formation of a

covalent bond from the 3’ to 5’ end 3’ to 5’ end of joining stands.

Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.

Lagging Strand

Okazaki Fragment 2Okazaki Fragment 2

DNA ligaseDNA ligase

Okazaki Fragment 1Okazaki Fragment 1

5’

5’

3’

3’



6.6. ProofreadingProofreading: initial base-pairing errors are usually corrected by DNA polymeraseDNA polymerase.


• Semiconservative Model:Semiconservative Model:

1. Watson and Crick showed:Watson and Crick showed: the two strands of the parental molecule separate, and

each functions as a template for synthesis of a new complementary strand.

Parental DNA

DNA Template

New DNA

DNA RepairDNA Repair

• Excision repair:Excision repair:

1. Damaged segment is excisedexcised by a repair repair enzymeenzyme (there are over 50 repair

enzymes).

2. DNA polymerase DNA polymerase and DNA ligase DNA ligase replace and bond the new nucleotides together.

Gene ExpressionTranscriptionTranslation

What is gene expression?

• Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product.

• A gene is expressed when its biological product is present and active.

• Gene expression is regulated at multiple levels.

Expression of Genetic Information

Beadle and Tatum (1941) showed in the fungus Neurospora crassa that there is a relationship

between a gene and each enzyme needed in a biochemical pathway, resulting in the one gene-

one enzyme hypothesis

(now modified to one gene-one polypeptide, since not all proteins are enzymes and some require

more than one polypeptide).


Production of proteins requires two steps:– Transcription involves an enzyme (RNA polymerase)

making an RNA copy of part of one DNA strand. There are four main classes of RNA:

i. Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic code.

ii. Transfer RNAs (tRNA).iii. Ribosomal RNAs (rRNA).iv. Small nuclear RNAs (snRNA), found only in eukaryotes.

– Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins.

RNA Synthesis DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer needed


Only some of the genes in a cell are active at any given time, and activity also varies by tissue type and developmental stage.

Regulation of gene expression is not completely understood, but it has been shown to involve an array of controlling signals.

a. Jacob and Monod (1961) proposed the operon model to explain prokaryotic gene regulation, showing that a genetic switch is used to control production of the enzymes needed to metabolize lactose. Similar systems control many genes in bacteria and their viruses.

b. Genetic switches used in eukaryotes are different and more complex, with much remaining to be learned about their function.

Protein Coding Genes

ORF– long (usually >100 aa)– “known” proteins likely

Basal signals– Transcription, translation

Regulatory signals– Depend on organism

•Prokaryotes vs Eukaryotes

Prokaryotic vs eukaryotic gene organization

1. Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site

2. Functionally related genes are frequently located near each other in prokayotes

3. These “operons” are transcribed into a single mRNA with internal translation initiation sites

Prokaryotic Gene Expression

Promoter Cistron1 Cistron2 CistronN Terminator

Transcription RNA Polymerase

mRNA 5’ 3’

TranslationRibosome, tRNAs,Protein Factors

1 2 N

Polypeptides

NC

NC N

C

1 2 3

Promoter Region on DNA

• upstream from transcription start site • initial binding site of RNA polymerase and initiation factors (IFs)• Promoter recognition: a prerequisite for initiation

E. coli consensus promoter regions

-10 site: “TATA” box

-35 site = TTGACA

(TATA box)

Bacterial operon

Eukaryotic genes

Eukaryotic gene organization

1.Transcripts begin and end beyond the coding region (5’UTR and 3’UTR)

2.The primary transcript is processed by:5’ capping3’ formation / polyA splicing

3.Mature transcripts are transported to the cytoplasm for translation

Regulation of gene expression

Plasmid

Gene (red) with an intron (green)Promoter

2. Transcription

Primary transcript

1. DNA replication

3. Posttranscriptional processing

4. Translation

mRNA degradation

Mature mRNA

5. Posttranslational processing

Protein degradationinactiveprotein

activeprotein

single copy vs. multicopy plasmids

Gene regulation of the transcription

Chr. I

Chr. II

Chr. III

Condition 1

“turned on”

“turned off”

Condition 2

“turned off”

“turned on”

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26

constitutively expressed gene

induced gene

repressedgene

inducible/ repressible genes

Gene regulation

constitutively expressed gene

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26

Condition 3 Condition 4 upregulated gene expression

down regulated gene expression

Definitions• Constitutively expressed genes

Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells.

• Inducible genesGenes that are transcribed and translated at higher levels in response to an inducing factor

• Repressible genesGenes whose transcription and translation decreases in response to a repressing signal

Definitions

• Housekeeping genes – genes for enzymes of central metabolic

pathways (e.g. TCA cycle)– these genes are constitutively expressed– the level of gene expression may vary

Modulators of transcription

• Modulators:(1) specificity factors, (2) repressors, (3) activators

1. Specificity factors:Alter the specificity of RNA polymerase

Examples: -factors (

70 32

Heat shock geneHousekeeping gene Heat shock promoter

Standard promoter


2. Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor binding

sites) Repressor binding is modulated by specific effectors

Coding sequence

Repressor

Operator

Promoter

Effector(e.g. endproduct)

Negative regulation

Repressor

EffectorExample: lac operon

RESULT:Transcription occurs when the gene is derepressed

Negative regulation

Repressor

Effector (= co-repressor)Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism


3. Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g.

enhancers) enhance the RNA polymerase -promoter interaction and

actively stimulate transcription common in eukaryotes

Coding sequence

Activator

promoter

RNA pol.

Positive regulation

RNA polymerase

Activator

Positive regulation

RNA polymerase

Activator Effector

Operons

a promoter plus a set of adjacent genes whose gene products function together.

usually contain 2 –6 genes, (up to 20 genes)these genes are transcribed as a

polycistronic transcript. relatively common in prokaryotes rare in eukaryotes

The lactose (lac) operon

• Contains several elements– lacZ gene = -galactosidase– lacY gene = galactosidase permease– lacA gene = thiogalactoside transacetylase– lacI gene = lac repressor

– Pi = promoter for the lacI gene– P = promoter for lac-operon– Q1 = main operator– Q2 and Q3 = secondary operator sites (pseudo-operators)

Pi P Z Y A I Q3 Q1 Q2

Regulation of the lac operon


Inducer molecules: Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside)- synthetic inducer, not metabolized,

lacI repressor


LacZ LacY LacA

Post-Transcriptional Modification in Eukaryotes

• primary transcript formed first• then processed (3 steps) to form mature mRNA • then transported to cytoplasm

Step 1: 7- methyl-guanosine “5’-cap” added to 5’ end

Step 2: introns spliced out; exons link up

Step 3: Poly-A tail added to 3’ end

mature mRNA5’-cap- exons -3’ PolyA tail

Intron Splicing in Eukaryotes

• Exons : coding regions• Introns: noncoding regions • Introns are removed by “splicing”

GUat 5’ endof intron

AGat 3’ endof intron

Splicesomes Roles in Spicing out Intron

RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes

Spliceosomes: protein + small RNAs (U1-8) complementary to the splice junctions

• 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome

• Exon termini are joined and sealed

Splicesomes Roles in Spicing out Intron

1 2

1 2

1 2

U1, U2 & U 5recognizedonor and acceptor sites

for splicingspecificity

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