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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