gene expression: transcription and translation, …blinderl/documents/transc_translpost.pdf · gene...
TRANSCRIPT
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Gene Expression: Transcription and Translation, Chapters 5 and 6
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Gene Expression
• Genes “control” phenotype
• Cellular environment affects gene expression
• Involves extensive intracellular signaling
• Tissue, development, disease specific
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THE CENTRAL DOGMA
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Which part is gene transcription? translation?
DNA RNA PROTEIN
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Transcription – Copy DNA to make mRNA
DNA + RNA polymerase + nucleotides mRNA template enzyme building blocks transcript
nucleotides in RNA _______
mRNA is ____ stranded
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Note that synthesis is 5’ -> 3’
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I. TRANSCRIPTION (prokaryotes)
A. INITIATION of Transcription
RNA polymerase recognizes gene promoter
holoenzyme
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Promoters = regulatory elements upstream of coding region of gene (+1) recognized by RNA polymerase
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Upstream Downstream
Prokaryotes: -10 box (Pribnow) and -35 box
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8 http://sandwalk.blogspot.com/2008/09/how-rna-
polymerase-binds-to-dna.html
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• Consensus sequence of promoters
• If promoter has a slightly different sequence, it will not be as “strong”
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TTGACA
TATAAT
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• sigma factor (s)of RNA pol binds promoter
• core polymerase locked on to form holoenzyme
• A variety of sigma factors
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The template strand of ds DNA is “read”
5’GGTATACCTACC3’ coding/sense 3’CCATATGGATGG5’ template
mRNA 5’G
RNA polymerase works 5’ 3’
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practice
5’ GTTAACCCATATGCTAGGGGG 3’ template strand
coding strand
mRNA transcript?
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TRANSCRIPTION: Initiation and Elongation
http://www.youtube.com/watch?v=41_Ne
5mS2ls&feature=related
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B. ELONGATION of mRNA transcript • RNA pol - synthesizes mRNA from template DNA
– DNA exposed one base at a time (transcription bubble)
– Template strand read to make mRNA
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http://www.scientificpsychic.com/fitness/transcription.gif
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Once RNA pol has moved, another RNA pol molecule can initiate to make another mRNA transcript. What does this mean?
Genetics institute – promoter and termination sites
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C. TERMINATION
Termination sequences
RNA pol loosens
mRNA dissociates
16 http://www.biog1105-1106.org/demos/106/unit02/media/termination-signal.jpg
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Polymerase fame
Severo Ochoa Arthur Kornberg
1/2 of the prize 1/2 of the prize
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II. Gene expression in eukaryotes
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List the differences
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contrast prokaryotes with eukaryotes
PROKARYOTES • no nucleus • 1 RNA polymerase • cotranslation • Polycistronic mRNA • One promoter shared by a few genes
• All of gene is coding = no modification of mRNA
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Eukaryotes - differences
1. 3 RNA polymerases RNA pol I – transcribes rRNA genes
RNA pol II – transcribes mRNA and snRNAs
RNA pol III – transcribes tRNA genes
2. Split genes
• Genes have coding and non-coding sequences
• Non-coding removed from mRNA
3. transcription and translation in separate compartments
4. Different regulatory elements 21
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Split genes in eukaryotes • exons - coding (expressed sequences)
• introns = intervening sequences
– non-coding but transcribed into mRNA
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Takifugu rubripes has few introns and far less repetitive DNA
400 million bp genome but same # genes as humans
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Split gene fame
• Philip Sharp and Richard Roberts (independent) Nobel 1993 for discovery of split genes
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Chick collagen = 37kb with 50 introns
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mRNA/DNA
hybrid
shows
introns as
loops
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Eukaryotic Promoters TATA promoter at -25
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Promoter proximal elements Basal element
Where is the transcription start site of the gene (base #1) ?
What are the black zig zag lines?
TATAAAA
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Promoter (s) = recognized by RNA polymerase II
UTR = untranslated region (transcribed, not translated)
Start – start site for transcription
Introns = non coding sequences within gene
Exons = coding regions
<- upstream downstream ->
+1
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4 types of genes • protein coding -> mRNAs -> protein
• tRNA genes -> transfer RNAs
• r RNA genes -> ribosomal RNAs
• snRNA genes -> small nuclear RNAs
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• TRANSCRIPTION OF PROTEIN CODING GENES
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Initiation of transcription in eukaryotes
2 types of proteins required:
1. Transcription factors allow RNA pol II to bind promoter
2. RNA polymerase II
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Stages of transcription
Initiation, Elongation, Termination
VIRTUAL CELL
http://vcell.ndsu.nodak.edu/animations/transcription/first.htm
TATA box, enhancer
TFs in general
RNA pol enzyme
mRNA Also http://www.youtube.com/watch?v=bk7PW1FKMTI
enhnacer, 7G, poly A tail, TF, spliceosome
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Pre-mRNA must be processed to mature mRNA
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Pre-mRNA is modified:
• At 5’ and 3’ ends
• Remove introns
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mRNA
DNA
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Intron to be removed from mRNA
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The European sea bass DAX1 gene structure
Martins et al. Reproductive Biology and Endocrinology 2007 5:19
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A . Addition of 7 methyl guanosine cap
• added to 5’ end of mRNA transcript
• increases half life of mRNA (stability)
• recognition site for ribosome binding
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A . Addition of 7 methyl guanosine cap
• Added to 5’ end of mRNA transcript
• increases half life of mRNA (stability)
• recognition site for ribosome binding
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B. Addition of the Poly A tail
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•Poly A tail of ~200 A’s added to end of transcript
•Increases the half life of the mRNA
Poly A tail
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Capping and tailing animation
• http://vcell.ndsu.nodak.edu/animations/mrnaprocessing/movie.htm
Transcription 7mG cap at 5’ end termination cleavage factors poly A tail at 3’ end splicing
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C. Removal of introns
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• What is the size in bases of the fully processed mature mRNA?
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b-globin gene
Expression of the b-globin gene is a typical process. This gene
contains two introns and three exons. Interestingly, the codon
of the 30th amino acid, AGG, is separated by an intron. As a
result, the first two nucleotides AG are in one exon and the third
nucleotide G is in another exon.
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• Splicing excises introns at specific exon/intron junction sites. Joins exons.
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GU……AG
Splice sites
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Intron information
• Variable in size
• May regulate gene expression
• Accumulate mutations
• Alternate splicing = more than one protein from a gene
• GU AG consensus sequences
• Some genes do not contain introns
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Intron removal
• http://vcell.ndsu.nodak.edu/animations/mrnasplicing/movie.htm
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Components of the spliceosome
• snRNPs + mRNA = spliceosome
“Snurps” are sn RNAs + proteins
– small nuclear RNAs (U1, U2, U4, U5, U6 ~ 100 bases)
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RNA world hypothesis
• RNA can possess enzymatic (protein) activity
• RNA-based life would be able to engage in enzymatic activities and store genetic information
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Gene mutations and beta thalassemia
• Beta thalassemia
– Mutation beta globin gene
– Inherited blood disorder (autosomal recessive)
– Can cause mild severe anemia, poor appetite, slow growth, bone abnormalities, enlarged heart
– Nucleated (immature) red blood cells
– More than 200 mutations identified
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About 20 new cases each year in US
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Thalassemia
• B0 no beta globin produced no Hb
• B+ low Hb production
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http://members.cox.net/amgough/Fanconi-genetics-genetics-
primer.htm#Nonsense
Single base change (point) mutations
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Single base change (point) mutations
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Stop codon
Frameshift mutation
Missense mutation
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Normal intron removal
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Loss of splice site
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Cis elements and trans factors
• Cis elements
– DNA sequences near gene that are required for gene expression
– Example:
• Trans factors – proteins that bind to DNA
– Example:
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DNA (cis factor) or protein (trans factor)?
Promoter
Transcription factors
Enhancer
TATA, CAAT, and GC
-10 and -35 box
RNA polymerase II
Which are found in prokaryotes? Eukaryotes? Both?
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VII. TRANSLATION
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mRNA protein
Nucleotides amino acids
Translation
http://vcell.ndsu.nodak.edu/animations/translation/movie.htm
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Translation end product =
PROTEINS
a. Amino acid – building block of protein
• 20 in nature
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b. Peptide – short stretch of amino acids
c. Polypeptide – the fully translated message
d. Protein – functional, 3D shape
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The genetic code
• Nirenberg and Khorana 1965 Nobel Prize
1. 4 nucleotide bases encode 20aa
TRIPLET CODE
64 possible codons
handout
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What is this amino acid sequence?
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2. Non-overlapping
3. Degenerate
More than 1 codon per particular amino acid
4. Universal
A few differences in mitochondria, yeast
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• Most polymorphisms are in the 3rd position of codon
– Why?
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5. 61 sense codons
start codon AUG (methionine)
• Initiation of translation
• All proteins start with met, in some removed post translation
6. 3 stop codons UAA, UAG, UGA
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Which part is the 5’ UTR of the mRNA?
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Example of the genetic code A partial mRNA is as follows:
5’ AGGAGGCUCGAACAUGUCAAUAUGCUUGUCCUGACGC 3’
• start site for translation?
• open reading frame?
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C. Ribosomes translate the mRNA to amino acid sequence
• 100,000s /cell
• protein + rRNA
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D. Translation steps
1. Initiation • mRNA read 5’ -> 3’ initiating at first AUG
• IFs involved
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2. Elongation
• “Charged” tRNAs bring amino acid to ribosome
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5’ Anticodon: 3' CGG 5'
Codon: 5' GCC 3‘
tRNA cloverleaf
structure – how?
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•2 codons can “sit” in a ribosome at once
Peptide bond between aa
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• wobble ->30 tRNAs for 61 codons
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DNA coding 5’ATG ACT AGC TGG GGG ATG TAC TTT TAG 3’
DNA template
mRNA
tRNA
anticodon
aa
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3. Termination of translation
• Stop codon
• RFs
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Polysome = many ribosomes can translate a
single message to produce many copies of a
protein
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• polysomes
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2. Protein structure
Primary structure –sequence of aa
• Secondary structure – spontaneous folds
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• Tertiary structure
– 3D
• Quaternary structure
– Some proteins have multiple subunits
– Example: hemoglobin
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Try it
• Translate a sequence Utah
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Translation tool
http://www.attotron.com/cybertory/analysis/trans.htm
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The anticodon is 5’ ACG 3’
What is the codon and the amino acid coded for?
The anticodon is 3’ GGC 5’
Amino acid?
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Codon CGU
Amino acid arginine
Codon CCG
Amino acid alanine
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VIII. Post translational modification - to the endoplasmic reticulum for processing
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IX. CONTROL OF GENE EXPRESSION
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1.Hormonal control of gene expression
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3. Splicing of mRNA
alternate splicing to form protein isoforms
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The fetal acetylcholine receptor, has variable kinetics during postnatal development. RT-
PCR revealed, in addition to the full-length mRNA, three new forms lacking exon 4.
One also lacks 19 nucleotides from exon 5, with a 43 residues shorter N-terminus.
A third one lacking the complete exon 5 predicts a subunit without transmembrane
segments.
These forms, generated by alternative splicing, may account for the kinetic variability of the
acetylcholine receptor channel.
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4. mRNA half life
poly A tail and 5’ cap. The longer the mRNA is in the cytoplasm, the more protein
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5. RNA interference (RNAi)
• Silencing of mRNA by tiny homologous dsRNA
• mRNA degradation
• RNAi can selectively turn off gene expression
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RNAi –dsRNA complementary to red eye pigment mRNA microinjected into Drosophila embryos
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• 6. Promoter strength – Number of transcripts made
end
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