510716- Gene Expression_52new

Usanee Anukool (Ph.D.) Clinical Microbiology Faculty of Associated Medical Sciences Chiang Mai University 2 July 2009

Aims After class, students will be able to:



Understand the principle of gene expression



Explain process of gene expression at the level of transcription and translation



Identify the differences of prokaryotic and eukaryotic transcription and ttranslation

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Contents Introduction to expression of genetic

information :

 The central dogma in molecular biology

Gene expression at the transcription level  Transcription in prokaryotes  Transcription in eukaryotes

Gene expression at the translation level  Translation in prokaryotes  Translation in eukaryotes

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Expression of genetic information The phenotype of an organism: Its genes & environmental factors Table 1. Genome sizes of different

species Species

Number of genes

Phage MS2

4

Phage T4

200

E. coli

4,000

Human

30,000-40,000 4

Expression of genetic information How do DNAs transmit

genetic information from generation to generation? DNA DNA  DNA replication 

How do the genetic

information is transferred into phenotype of an organism? DNA  Protein  Transcription to translation 

The process of gene expression The Central Dogma Reverse transcription

DNA Protein Transcription

RNA Translation

Replication

Regulations 6

Transcription & Translation Transcription The synthesis of an RNA transcript

complementary to one strand of DNA in gene

Translation The conversion of genetic information stored

in the nucleotide sequence in RNA transcript into amino acid sequence of polypeptides

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Transcription RNA polymerase DNA

RNA molecules mRNA tRNA rRNA snRNA

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RNA molecules messenger RNA: mRNAs The intermediaries that carry genetic information from DNA to ribosomes when protein are synthesized transfer RNA: tRNA Small RNA molecule that function as adaptors between amino acids and codons in mRNA during translation

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RNA molecules ribosomal RNA: rRNA Structural and catalytic components of ribosomes  Ribosomes, the intricate machines that translate nucleotide sequence of mRNAs into amino acid sequence of polypeptides small nuclear RNA: snRNA Structural components of spliceosomes  Spliceosomes, the nuclear structure excise introns from nuclear genes

that 10

Transcription The mechanism is similar to that of DNA

replication Excepted that: The precursors are ribonucleotide Only one strand of DNA is used as a ‘template

strand’ No requirement of primer

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Transcription The RNA chain synthesized is Complementary to the ‘template strand’ Identical to the ‘nontemplate strand’ ‘Sense strand’: an mRNA molecule/ the

coding strand of RNA ‘Antisense strand’: an RNA molecule that is complementary to the mRNA strand

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Transcription

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Transcription In General Process: The transcription stages 2.Initiation 3.Elongation 4.Termination

Key enzyme: DNA-dependent RNA Polymerase 14

Transcription The RNA polymerase Initiate at a specific nucleotide sequence

called ‘promoter’ Catalyze the RNA chain elongation (5’ 3’) DNA template

n(RTP)

RNA polymerase

(RMP)n + n(PP)

RTP = ribonucleotide triphosphate  RMP = ribonucleotide monophosphate 

RNA:

U replaces T (U=A, C≡G)

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Transcription in Prokaryotes RNA polymerase Only one polymerase required in prokaryotes Mutimeric proteins (MW~480,000 Da) Holoenzyme: 5 polypeptides (2α,β, β’,σ)  Two are identical (α)  Thus contains 4 distinct polypeptides with different functions Core enzyme: (2α,β, β’) : involved both

initiation and elongation steps The sigma (σ) factor: involved in the initiation only

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Transcription in Prokaryotes The sigma (σ ) factor Recognize and bind RNA polymerase to the transcription initiation/ promoter site in DNA in vitro experiment:  Without sigma factor (2α,β, β’), RNA chain initiates randomly in DNA sequence

sigma factor (2α,β, β’,σ ), RNA chain initiates specifically at sites used in vivo

 With

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Transcription in Prokaryotes  Initiation of RNA chain

Binding of RNA pol. to a promoter regions in DNA :Two short conserved regions:  

The -10 sequence: TATAAT box The -35 sequence: TTGACA box  The recognition sequence where the sigma factor initially recognizes and binds to

Localised unwinding of the dsDNA by RNA pol. Formation of phosphodiester bonds between the first few ribo-nts in nascent RNA 18 chain

•Structure of typical promoter in E.coli

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Transcription in Prokaryotes Elongation of RNA chain Extension of RNA chain Catalyzed by RNA polymerase core enzyme after release of σ factor Occur at the ‘transcription bubble’ Unwinding and rewinding by RNA pol. activities In E. coli:  average length of a transcription bubble is 18 nt, and about 40 ribo-nt are incorporated into the growing chain per second

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•Elongation of RNA chain in E.coli

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Transcription in Prokaryotes Termination of RNA chain

The transcription complex dissociated,

releasing nascent RNA

Occur when RNA pol encounters a special

termination signal called “transcription terminator”

Two types of transcription terminators in E.

coli

 rho 

Require a presence of rho protein

 rho 

(ρ)−dependent terminators (ρ)− independent terminators

The sequence contain GC-rich region followed by 6 AT bp 22

•Structure of Rho-independent transcription terminators DNA

RNA

Folded RNA

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Transcription in Eukaryotes More complex process with three classes of RNA

pol.

Enzymes

location

Products

Sensitivity to α-amanitin

RNA pol. I

nucleolus rRNAs, excluding 5S-rRNA

No

RNA pol. II

nucleus

Nuclear pre-mRNAs

complete

RNA pol. III

nucleus

tRNAs, 5s-rRNA, snRNAS

Intermediate

All three RNA pol. requires the assistance of other

proteins called transcription factors

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Transcription in Eukaryotes  The majority of the primary transcripts

undergo three major modifications prior to their transport to the cytoplasm: 1. Addition of 7-methyl guanosine caps at the 5’end 2. Addition Poly(A) tail at the 3’ end 3. Splicing of intron sequence

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•Posttranscriptional processing of RNA transcript in eukaryotes

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Transcription in Eukaryotes Initiation of RNA chain Initiation complex RNA pol.  Promoter region in RNA  Transcription factors (TFs) 

RNA pol II’s promoter region: short highly conserved

modules , “the TATA box binding protein, TBP” TATA box : TATAAAA (centered at -30)  CAAT box : GGCCAATCT (near -80) 

TFII-D, A, B, F, E 27

•Initiation of transcription by RNA pol. II

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Transcription in Eukaryotes RNA chain elongation Similar to that in prokaryotes But using three different enzymes:  RNA

Pol I; II & III and addition of 7-methylguanosine (7-MG) caps at 5’- end (occur when about 30 nt chain long)

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•Addition of 7-methyl-guanosine caps at 5’-end

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Transcription in Eukaryotes Termination Chain cleavage: occur at downstream

from a polyadenylation signal ‘AAUAAA’ by endonucleolytic activity Polyadenylation is catalysed by

poly(A) Polymerase Poly(A) tail (~ 200 nt long) 31

Polyadenylation

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RNA splicing

Removal of intron sequence Acuracy: exon-GT..…AG-exon (conserved dinucleotide sequence)

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RNA Splicing Three types of intron excision Intron of tRNA precursor  Endonuclease & ligase activity Intron of rRNA precursor  Automatic cleavage (no enzyme involvement) Intron of mRNA precursor  carried out by complex ribonucleoprotein particles called ‘Spliceosomes’  Spliceosomes: snRNAs-protein complex 34

Splicing of tRNA precursor

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Splicing of rRNA precursor

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Splicing of mRNA precursor

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Translation Protein 15% of wet wt. of cells Play many roles vital to the lives of all cells Polypeptides: 20 different amino acids Amino acids: H H O Carboxyl group Amino group H-N - C - C - OH R Side chain 38

•The formation of a peptide bond and four levels of protein organization

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The Genetic Code Codon = a unit or word specify one amino acid or, actually, one aminoacyl-tRNA

Triplets Non-overlapping  except rare case: nt are read in 2 different directions

Comma-free Degenerate:  Each amino acid is coded by one or more codons  20 amino acids: 4 different nucleotides, 61 codons

Ordered, usually differing by a single nucleotide Contains start and stop codons  AUG (GUG), UAG/UGA/UAA

Nearly universal  (with

minor organisms

exception)

same

meaning

in

all 40

•The genetic codes

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•Overview of protein synthesis

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Translation Occurs

on ribosomes located cytoplasm Involves 3 types of RNAs

in

the

mRNA, 3-5 rRNA, 40-60 tRNA molecules

tRNA are activated by an aminoacyl tRNA

synthetases

act as adaptor mediating the incorporation

of proper amino acids to polypeptides

Polyribosomes: each mRNA is simultaneously translated by several ribosomes 43

Translation components Ribosomes rRNA-protein complex macromolecules Two subunits: Large & Small rRNA synthesis (RNA pol. I) and

ribosomes assembly occur in the nucleolus

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•Composition of prokaryotic and eukaryotic ribosomes

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Translation components tRNAs Amino acid attach to tRNA by high energy

bond between carboxyl group of amino acid and 3’-hydroxyl termini of tRNA Aminoacyl-tRNA synthetase activated and charged tRNAs with amino acids in two steps: 1. aa + ATP 2. aa-AMP + tRNA

aa-AMP + Ppi aa-tRNA + AMP 46

•The tRNA molecules Nucleotide sequence and cloverleaf configuration

Molecular model of yeast phenylalanine tRNA based on x-ray diffraction data

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Translation  The three stages of translation 1. Polypeptide chain initiation 2. Chain elongation 3. Chain termination

 The initiation of translation 

Similar between prokaryotes and eukaryote in many aspects but there are some differences 48

Translation The initiation of translation in E. coli

requires: 30S subunit of the ribosome A special initiator tRNA  tRNA Met

: the translation initiation codon (AUG/GUG)  carries formyl-Methionine f

An mRNA molecule Three soluble protein initiation factors: IF-1, IF-

2, and IF-3 One molecule of GTP Initiation sequence: “Shine-Dalgarno

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•The initiation translation in E. coli

Shine-Dalgarno sequence mRNA

Start codon

5’...AAC [AGGAGG] AUAUCC AUG UC…3’

16S rRNA 3’…AUGAGAU [UCCUCC] ACUAGG……..5’ Region of base pairing

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•The initiation translation in E. coli

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Translation The

initiation of translation eukaryotes Similar process excepted that: More

in

complex with many soluble initiation

factors tRNAiMet the methionine is not formylated, i=initiator)  enter the P site directly (same as in E. coli) 

Initiation complex form at 5’ terminus of mRNA,

not at Shine-Dalgarno sequences (Randomly scan for start codon)

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Translation The

initiation of eukaryotes (more)

translation

in

A cap-binding protein (CBP) binds to the 7-

methyl G cap at 5’ terminus in mRNA then other Ifs bind to CBP-mRNA complex followed by small (40S) subunit When AUG is found, IFs are dissociated from

the complex and large subunit (60S) bind to methyonyl-tRNA/mRNA/40S complex 53

Translation 

The elongation of translation : basically the same both in prokaryotes and eukaryotes (similar elongation factors : EF)



Three steps of elongation (repeated in cyclic maner) • Binding of aminoacyl-tRNA to the A site of the ribosome (EF-Tu/GTP, EF-Ts) • Transfer of growing peptide chain from tRNA from the P site to A site by peptide bond formation catalysed by peptidyl transferase • Translocation of ribosome along the mRNA

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•Peptide chain elongation in E. coli

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Translation  The 



 In 

 In 

termination of translation

Occur when the chain termination codons: UAA, UAG, or UGA enter A site on the ribosome Recognized by soluble protein called release factors (RFs)

E. coli : RF-1 (UAA, UAG) and RF-2 (UAA, UGA)

eukaryotes: Single RF (eRF) recognizes all three termination codons

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•Polypeptide chain termination in E. coli

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Further readings 



Snustad, D.P., and Simmons, M.J. 2003. In Principle of Genetics, 3rd ed., pp279-326. John Willy & Sons, Inc., USA. Textbooks in Molecular Genetics, The Cell & Molecular Biology or Biochemistry

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http://www.web-books.com/MoBio/Free/Ch4E.htm 59

3’

5’ Enhancer (-1000s)

Promoter site (TBP, -30 and -80)

upstream control elements (UCEs) (-200 bases)

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