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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

2

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

3

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

7

Transcription RNA polymerase DNA

RNA molecules mRNA tRNA rRNA snRNA

8

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

9

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

11

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

12

Transcription

13

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)

15

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

16

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

17

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

19

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

20

•Elongation of RNA chain in E.coli

21

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

23

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

24

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

25

•Posttranscriptional processing of RNA transcript in eukaryotes

26

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

28

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)

29

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

30

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

32

RNA splicing

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

33

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

35

Splicing of rRNA precursor

36

Splicing of mRNA precursor

37

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

39

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

41

•Overview of protein synthesis

42

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

44

•Composition of prokaryotic and eukaryotic ribosomes

45

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

47

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

49

•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

50

•The initiation translation in E. coli

51

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)

52

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

54

•Peptide chain elongation in E. coli

55

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

56

•Polypeptide chain termination in E. coli

57

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

58

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)

60

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