Chapter 17~ From Gene to Protein

1
Chapter 17 From Gene to Protein
2
Protein Synthesis overview

• One gene-one enzyme hypothesis (Beadle and Tatum)
• One gene-one polypeptide (protein) hypothesis
• Transcription synthesis of RNA under the
  direction of DNA (mRNA)
• Translation actual synthesis of
  a polypeptide under the direction of mRNA

3
The Central Dogma

• 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
4
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
5
Transcription

• fromDNA nucleic acid languagetoRNA nucleic
  acid language

6
RNA

• ribose sugar
• N-bases
• uracil instead of thymine
• U A
• C G
• single stranded
• lots of RNAs
• mRNA, tRNA, rRNA, siRNA

transcription
RNA
DNA
7
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?
8
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

9
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

10
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

11
Matching bases of DNA RNA
A

• Match RNA bases to DNA bases on one of the DNA
  strands

C
U
G
A
G
G
U
C
U
U
G
C
A
C
A
U
A
G
A
C
U
A
5'
3'
G
A
C
C
A
G
G
G
G
G
G
T
T
A
C
A
C
T
T
T
T
T
C
C
C
C
A
A
12
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

13
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
14
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
15
Discovery of exons/introns
1977 1993
Richard Roberts
Philip Sharp
adenovirus
CSHL
MIT
common cold
beta-thalassemia
16
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
17
RNA splicing enzymes
Whoa! I think we just brokea biological rule!

• snRNPs
• small nuclear RNA
• proteins
• Spliceosome
• several snRNPs
• recognize splice site sequence
• cut paste gene

No, not smurfs! snurps
18
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!
19
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

20
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
21
Translation

• fromnucleic acid languagetoamino acid language

22
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)?

23
mRNA codes for proteins in triplets
24
Cracking the code
1960 1968
Nirenberg Khorana

• Crick
• determined 3-letter (triplet) codon system

WHYDIDTHEREDBATEATTHEFATRAT
WHYDIDTHEREDBATEATTHEFATRAT

• Nirenberg (47) Khorana (17)
• 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

25
1960 1968
Marshall Nirenberg
Har Khorana
26
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

27
How are the codons matched to amino acids?
3?
5?
TACGCACATTTACGTACGCGG
DNA
5?
3?
AUGCGUGUAAAUGCAUGCGCC
mRNA
codon
3?
5?
tRNA
anti-codon
aminoacid
28
From gene to protein
nucleus
cytoplasm
transcription
translation
mRNA
DNA
protein
trait
29
Transfer RNA structure

• Clover leaf structure
• anticodon on clover leaf end
• amino acid attached on 3? end

30
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 condon of mRNA
31
Ribosomes

• Facilitate coupling of tRNA anticodon to mRNA
  codon
• organelle or enzyme?
• Structure
• ribosomal RNA (rRNA) proteins
• 2 subunits
• large
• small

E
P
A
32
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
33
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
34
Protein targeting

• Destinations
• secretion
• nucleus
• mitochondria
• chloroplasts
• cell membrane
• cytoplasm
• etc

• Signal peptide
• address label

start of a secretory pathway
35
RNA polymerase
DNA
Can you tell the story?
aminoacids
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
aminoacyl tRNAsynthetase
poly-A tail
3'
large ribosomal subunit
polypeptide
5'
tRNA
small ribosomal subunit
E
P
A
ribosome
36
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
37
Translation in Prokaryotes

• Transcription translation are simultaneous in
  bacteria
• DNA is in cytoplasm
• no mRNA editing
• ribosomes read mRNA as it is being transcribed
  

38
Translation prokaryotes vs. eukaryotes

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

39
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?
40
Point mutation leads to Sickle cell anemia

• What kind of mutation?

Missense!
41
Sickle cell anemia

• Primarily Africans
• recessive inheritance pattern
• strikes 1 out of 400 African Americans

hydrophilicamino acid
hydrophobic amino acid
42
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?
43
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

44
Deletion leads to Cystic fibrosis
delta F508
loss of oneamino acid
45
Whats the value ofmutations?