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Ch. 17 Lecture

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Ch. 17 Lecture Flow of genetic information in a cell How do we move information from DNA to proteins? transcription translation RNA protein DNA trait – PowerPoint PPT presentation

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Title: Ch. 17 Lecture


1
Ch. 17 Lecture
  • Flow of genetic information in a cell
  • How do we move information from DNA to proteins?

transcription
translation
protein
RNA
DNA
trait
DNA gets all the glory, but proteins do all
the work!
replication
2
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
3
RNA
  • ribose sugar
  • N-bases
  • uracil instead of thymine
  • U A
  • C G
  • single stranded
  • lots of RNAs
  • mRNA, tRNA, rRNA, snRNA

transcription
RNA
DNA
4
Transcription
  • Making mRNA
  • transcribed DNA strand template strand
  • untranscribed DNA strand coding strand
  • same sequence as RNA
  • synthesis of complementary RNA strand
  • transcription bubble
  • enzyme
  • RNA polymerase

coding strand
3?
C
C
G
G
A
A
T
T
5?
A
G
A
A
A
C
G
T
T
T
T
C
A
T
C
G
C
A
T
DNA
3?
C
T
G
A
A
5?
T
G
C
C
G
G
A
U
U
T
C
unwinding
3?
C
G
G
A
A
T
rewinding
mRNA
template strand
RNA polymerase
5?
build RNA 5??3?
5
RNA polymerases
  • 3 RNA polymerase enzymes
  • RNA polymerase 1
  • only transcribes rRNA genes
  • makes ribosomes
  • RNA polymerase 2
  • transcribes genes into mRNA
  • RNA polymerase 3
  • only transcribes tRNA genes
  • each has a specific promoter sequence it
    recognizes

6
Which gene is read?
  • Promoter region
  • binding site before beginning of gene
  • TATA box binding site
  • binding site for RNA polymerase transcription
    factors
  • Enhancer region
  • binding site far upstream of gene
  • turns transcription on HIGH

7
Transcription Factors
  • Initiation complex
  • transcription factors bind to promoter region
  • suite of proteins which bind to DNA
  • hormones
  • turn on or off transcription
  • trigger the binding of RNA polymerase to DNA

8
Transcription the process
  • 1.Initiation transcription factors mediate the
    binding of RNA polymerase to an initiation
    sequence (TATA box)
  • 2.Elongation RNA polymerase continues unwinding
    DNA and adding nucleotides to the 3 end
  • 3.Termination RNA polymerase reaches terminator
    sequence

9
Eukaryotic genes have junk!
  • Eukaryotic genes are not continuous
  • exons the real gene
  • expressed / coding DNA
  • introns the junk
  • inbetween sequence

intronscome out!
eukaryotic DNA
10
mRNA splicing
  • Post-transcriptional processing
  • eukaryotic mRNA needs work after transcription
  • primary transcript pre-mRNA
  • mRNA splicing
  • edit out introns
  • make mature mRNA transcript

10,000 bases
eukaryotic DNA
pre-mRNA
primary mRNA transcript
1,000 bases
mature mRNA transcript
spliced mRNA
11
Splicing must be accurate
  • No room for mistakes!
  • a single base added or lost throws off the
    reading frame

AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
MetArgSerAspLysGlyHis
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
MetArgValArgSTOP
12
RNA splicing enzymes
  • snRNPs
  • small nuclear RNA
  • proteins
  • Spliceosome
  • several snRNPs
  • recognize splice site sequence
  • cut paste gene

No, not smurfs! snurps
13
Alternative splicing
  • Alternative mRNAs produced from same gene
  • when is an intron not an intron
  • different segments treated as exons

Starting to gethard to define a gene!
14
More post-transcriptional processing
  • Need to protect mRNA on its trip from nucleus to
    cytoplasm
  • enzymes in cytoplasm attack mRNA
  • protect the ends of the molecule
  • add 5? GTP cap
  • add poly-A tail
  • longer tail, mRNA lasts longer produces more
    protein

15
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
16
How does mRNA code for proteins?
4
ATCG
4
AUCG
20
  • How can you code for 20 amino acids with only 4
    nucleotide bases (A,U,G,C)?

17
mRNA codes for proteins in triplets
18
Cracking the code
1960 1968
Nirenberg Khorana
  • Crick
  • determined 3-letter (triplet) codon system

WHYDIDTHEREDBATEATTHEFATRAT
WHYDIDTHEREDBATEATTHEFATRAT
  • Nirenberg Khorana
  • determined mRNAamino acid match
  • added fabricated mRNA to test tube of ribosomes,
    tRNA amino acids
  • created artificial UUUUU mRNA
  • found that UUU coded for phenylalanine

19
1960 1968
Marshall Nirenberg
Har Khorana
20
The code
  • Code for ALL life!
  • strongest support for a common origin for all
    life
  • Code is redundant
  • several codons for each amino acid
  • 3rd base wobble

Why is thewobble good?
  • Start codon
  • AUG
  • methionine
  • Stop codons
  • UGA, UAA, UAG

21
How are the codons matched to amino acids?
3?
5?
TACGCACATTTACGTACGCGG
DNA
5?
3?
AUGCGUGUAAAUGCAUGCGCC
mRNA
codon
3?
5?
tRNA
anti-codon
aminoacid
22
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
23
Transfer RNA structure
  • Clover leaf structure
  • anticodon on clover leaf end
  • amino acid attached on 3? end

24
Loading tRNA
  • Aminoacyl tRNA synthetase
  • enzyme which bonds amino acid to tRNA
  • bond requires energy
  • ATP ? AMP
  • bond is unstable
  • so it can release amino acid at ribosome easily

Trp
CO
Trp
Trp
CO
H2O
OH
O
OH
CO
O
activating enzyme
tRNATrp
A
C
C
mRNA
U
G
G
anticodon
tryptophan attached to tRNATrp
tRNATrp binds to UGG codon of mRNA
25
Ribosomes
  • Facilitate coupling of tRNA anticodon to mRNA
    codon
  • organelle or enzyme?
  • Structure
  • ribosomal RNA (rRNA) proteins
  • 2 subunits
  • large
  • small

E
P
A
26
Ribosomes
  • A site (aminoacyl-tRNA site)
  • holds tRNA carrying next amino acid to be added
    to chain
  • P site (peptidyl-tRNA site)
  • holds tRNA carrying growing polypeptide chain
  • E site (exit site)
  • empty tRNA leaves ribosome from exit site

Met
C
A
U
5'
G
U
A
3'
A
P
E
27
Building a polypeptide
  • Initiation
  • brings together mRNA, ribosome subunits,
    initiator tRNA
  • Elongation
  • adding amino acids based on codon sequence
  • Termination
  • end codon

release factor
Leu
Val
Ser
Met
Met
Ala
Leu
Met
Met
Leu
Leu
Trp
tRNA
C
A
G
C
A
G
C
C
A
A
C
G
U
A
C
G
C
A
C
U
A
U
A
U
U
A
5'
5'
A
A
A
G
5'
U
C
U
A
5'
G
G
A
A
A
G
U
U
U
C
U
G
G
U
U
3'
C
A
U
C
G
G
A
U
A
U
A
A
C
C
mRNA
3'
3'
3'
A
A
U
U
G
G
3'
P
E
A
28
Protein targeting
  • Destinations
  • secretion
  • nucleus
  • mitochondria
  • chloroplasts
  • cell membrane
  • cytoplasm
  • etc
  • Signal peptide
  • address label

start of a secretory pathway
29
RNA polymerase
DNA
Can you tell the story?
aminoacids
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
aminoacyl tRNAsynthetase
poly-A tail
large ribosomal subunit
3'
polypeptide
5'
tRNA
small ribosomal subunit
E
P
A
ribosome
30
Prokaryote vs. Eukaryote genes
  • Prokaryotes
  • DNA in cytoplasm
  • circular chromosome
  • naked DNA
  • no introns
  • Eukaryotes
  • DNA in nucleus
  • linear chromosomes
  • DNA wound on histone proteins
  • introns vs. exons

intronscome out!
eukaryotic DNA
31
Translation in Prokaryotes
  • Transcription translation are simultaneous in
    bacteria
  • DNA is in cytoplasm
  • no mRNA editing
  • ribosomes read mRNA as it is being transcribed

32
Translation prokaryotes vs. eukaryotes
  • Differences between prokaryotes eukaryotes
  • time physical separation between processes
  • takes eukaryote 1 hour from DNA to protein
  • no RNA processing

33
Mutations
  • Point mutations
  • single base change
  • base-pair substitution
  • silent mutation
  • no amino acid change
  • redundancy in code
  • missense
  • change amino acid
  • nonsense
  • change to stop codon

When do mutationsaffect the nextgeneration?
34
Point mutation leads to Sickle cell anemia
  • What kind of mutation?

Missense!
35
Sickle cell anemia
  • Primarily Africans
  • recessive inheritance pattern
  • strikes 1 out of 400 African Americans

hydrophilicamino acid
hydrophobic amino acid
36
Mutations
  • Frameshift
  • shift in the reading frame
  • changes everything downstream
  • insertions
  • adding base(s)
  • deletions
  • losing base(s)

Where would this mutation cause the most
changebeginning or end of gene?
37
Cystic fibrosis
  • Primarily whites of European descent
  • strikes 1 in 2500 births
  • 1 in 25 whites is a carrier (Aa)
  • normal allele codes for a membrane protein that
    transports Cl- across cell membrane
  • defective or absent channels limit transport of
    Cl- ( H2O) across cell membrane
  • thicker stickier mucus coats around cells
  • mucus build-up in the pancreas, lungs, digestive
    tract causes bacterial infections
  • without treatment children die before 5 with
    treatment can live past their late 20s

38
Deletion leads to Cystic fibrosis
delta F508
loss of oneamino acid
39
Whats the value ofmutations?
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