Download DNA to Protein

Chapter 14
From Gene
to Protein
Metabolism Teaches Us About Genes

Metabolic defects

studying metabolic diseases suggested
that genes specified proteins



A
alkaptonuria (black urine from alkapton
a.k.a. homogentisic acid )
PKU (phenylketonuria)
Genes
each disease is caused by
non-functional enzyme
B
C
create
phenotype…
D
E
1 Gene – 1 Enzyme Hypothesis

Beadle & Tatum

Compared mutants of bread mold,
Neurospora fungus

created mutations by X-ray treatments
 X-rays break DNA
 inactivate a gene

wild type grows on “minimal” media
 sugars + required precursor nutrient to synthesize
essential amino acids

mutants require added amino acids
 each type of mutant lacks a certain enzyme
needed to produce a certain amino acid
 non-functional enzyme = broken gene
1941 | 1958
Beadle & Tatum
George Beadle
Edward Tatum
Beadle & Tatum’s Neurospora
Experiment
So… What is a Gene?

One gene – one enzyme



One gene – one protein



each protein has its own gene
but many proteins are composed of several
polypeptides
One gene – one polypeptide


all proteins are coded by genes
but not all proteins are enzymes
but many genes only code for RNA
One gene – one product

but many genes code for
more than one product …
Where
does that
leave
us?!
Defining a Gene…
“Defining a gene is problematic because…
one gene can code for several protein
products, some genes code only for RNA,
two genes can overlap, and there are many
other complications.”
– Elizabeth Pennisi, Science 2003
gene
RNA
polypeptide 1
gene
polypeptide 2
polypeptide 3
It’s hard to
hunt for wabbits,
if you don’t know
what a wabbit
looks like.
The “Central Dogma”

How do we move information from
DNA to proteins?
transcription
DNA
replication
translation
RNA
protein
For
simplicity sake,
let’s go back to
genes that code
for proteins…
From nucleus to cytoplasm…

Where are the genes?


Where are proteins synthesized?


genes are on chromosomes in nucleus
proteins made in cytoplasm by ribosomes
How does the information get from
nucleus to cytoplasm?

messenger RNA
nucleus
RNA


ribose sugar
N-bases
uracil instead of thymine
U : A
C : G



single stranded
mRNA, rRNA, tRNA,
siRNA….
transcription
DNA
RNA
Transcription

Transcribed DNA strand = template strand


Synthesis of complementary RNA strand


untranscribed DNA strand = coding strand
transcription bubble
Enzyme that facilitates the building of RNA:

RNA polymerase
Transcription

Initiation

RNA polymerase binds to promoter
sequence on DNA
Role of promoter
1. Where to start reading
= starting point
2. Which strand to read
= template strand
3. Direction on DNA
= always reads DNA
3'5'
Transcription

Elongation
RNA polymerase unwinds DNA
~20 base pairs at a time
 reads DNA 3’5’
 builds RNA 5’3’ (the enzyme governs the synthesis!)

No proofreading
1 error/105 bases
 many copies
 short life
 not worth it!

Transcription
RNA
Transcription

Termination
RNA polymerase stops at termination
sequence
 mRNA leaves nucleus through pores

RNA GC
hairpin turn
Prokaryote vs. Eukaryote Genetics

Differences between
prokaryotes & eukaryotes
time & physical separation
between processes
 RNA processing

Transcription in Eukaryotes


only 1 prokaryotic enzyme
3 eukaryotic RNA polymerase enzymes

RNA polymerase I


RNA polymerase I I


transcribes genes into mRNA
RNA polymerase I I I


only transcribes rRNA genes
only transcribes rRNA genes
each has a specific promoter sequence
it recognizes
Eukaryotic Post-transcriptional Processing

Primary transcript


eukaryotic mRNA needs work after transcription
Protect mRNA

from RNA-ase enzymes in cytoplasm
 add 5' G cap
mRNA
5' cap
 add polyA tail 5' G PPP
CH
3'
A
3

Edit out introns
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
primary mRNA
transcript
mature mRNA
transcript
pre-mRNA
spliced mRNA
Primary Transcript

Processing mRNA

protecting RNA from RNase in cytoplasm



add 5’ cap
add polyA tail
remove introns
AUG
UGA
Protecting RNA

5’ cap added


G trinucleoside (G-P-P-P)
protects mRNA


from RNase (hydrolytic enzymes)
3’ poly-A tail added


50-250 A’s
protects mRNA


from RNase (hydrolytic enzymes)
helps export of RNA from nucleus
UTR
UTR
Dicing & Splicing mRNA

Pre-mRNA  mRNA

edit out introns


splice together exons


intervening sequences
expressed sequences
“AVERAGE”
“gene” = 8000b
pre-mRNA = 8000b
mature mRNA = 1200b
protein = 400aa
lotsa “JUNK”!
In higher eukaryotes


90% or more of gene can be intron
no one knows why…yet
 there’s a Nobel prize waiting…
Splicing Enzymes

snRNPs



small nuclear RNA
RNA + proteins
Spliceosome


several snRNPs
recognize splice site
sequence


cut & paste
RNA as ribozyme


some mRNA can
splice itself
RNA as enzyme
1982 | 1989
Ribozyme

RNA as enzyme
Sidney Altman
Yale
Thomas Cech
U of Colorado
Splicing Details

No room for mistakes!
editing & splicing must be exactly accurate
 a single base added or lost throws off the
reading frame
AUGCGGCUAUGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met | Arg | Ser | Asp | Lys | Gly | His

AUGCGGCUAUGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met | Arg | Val | Arg |STOP|
Alternative Splicing

Alternative mRNAs produced from same gene


when is an intron not an intron…
different segments treated as exons
Hard
to define
a gene!
Discovery of Split Genes
Richard Roberts
NE BioLabs
Philip Sharp
MIT
1977 | 1993
adenovirus
common cold
Structure of Antibodies
Y
Y
Y
Y
s
s
s
light
chain
B cell
membrane
s
s
s
s
s s
s s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
Y
s
Y
s
s
s
Y
s
s
Y
s
variable region
s
Y
s
s
Y
s
Y
Y
antigen-binding site
light
chain
heavy
chains
light chains
antigen-binding
site
heavy chains
antigen-binding
site
How do vertebrates
produce millions of
antibody proteins, if
they only have a few
hundred genes coding
for those proteins?
By DNA rearrangement
& somatic mutation
vertebrates can
produce millions of
B & T cells
antibody
mRNA
DNA of differentiated B cell
C
chromosome of undifferentiated B cell
rearrangement
of DNA
V
D
C
J
B cell
The Transcriptional Unit (gene?)
enhancer
1000+b
20-30b
3'
RNA
TATA
polymerase
translation
start
TAC
translation
stop
exons
transcriptional unit
5'
DNA
ACT
DNA
UTR
promoter
UTR
introns
transcription
start
transcription
stop
5'
pre-mRNA
5'
GTP mature mRNA
3'
3'
AAAAAAAA
Prokaryote vs. Eukaryote Genetics

Prokaryotes

DNA in cytoplasm
 circular
chromosome
 naked DNA


no introns
Eukaryotes
DNA in nucleus
 linear
chromosomes
 DNA wound on
histone proteins
 introns vs. exons

intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
From Gene to Protein
aa
aa
aa
transcription
DNA
translation
aa
aa
mRNA
protein
aa
aa
aa
mRNA leaves
nucleus through
nuclear pores
nucleus
ribosome
cytoplasm
proteins synthesized
by ribosomes using
instructions on mRNA
Translation in Prokaryotes

Transcription & translation are
simultaneous in bacteria
DNA is in
cytoplasm
 no mRNA
editing
needed

How Does DNA Code for Proteins
DNA
TACGCACATTTACGTACGCGG
mRNA
AUGCGUGUAAAUGCAUGCGCC
?
protein Met Arg Val Asn Ala Cys Ala
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
Cracking the Code

1960 | 1968
Nirenberg & Matthaei

determined 1st codon–amino acid match

UUU coded for phenylalanine
created artificial poly(U) mRNA
 added mRNA to test tube of
ribosomes, tRNA & amino acids


mRNA synthesized single
amino acid polypeptide chain
phe–phe–phe–phe–phe–phe
Heinrich Matthaei
Marshall Nirenberg
Translation

Codons

blocks of 3
nucleotides
decoded into
the sequence
of amino acids
mRNA Codes for Proteins in Triplets
TACGCACATTTACGTACGCGG
DNA
codon
mRNA
AUGCGUGUAAAUGCAUGCGCC
?
protein
Met Arg Val Asn Ala Cys Ala
mRNA codes for proteins in triplets…
CODONS!
The Code!

For ALL life!


strongest support
for a common origin
for all life
Code is redundant

several codons for
each amino acid
Why is this a good thing?

Start codon



AUG
methionine
Stop codons

UGA, UAA, UAG
How are Codons Matched to
Amino Acids?
3'
DNA
5'
TACGCACATTTACGTACGCGG
5'
mRNA
3'
AUGCGUGUAAAUGCAUGCGCC
UAC GCA
3'
5'
tRNA
amino
acid
Met Arg
codon
CAU
Val
anti-codon
aa
aa
aa
cytoplasm
transcription
translation
aa
aa
aa
aa
aa
protein
aa
aa
aa
nucleus
tRNA Structure

“Clover leaf” structure
anticodon on “clover leaf” end
 amino acid attached on 3' end

Loading tRNA

Aminoacyl tRNA synthetase
enzyme which bonds
amino acid to tRNA
 endergonic reaction



ATP  AMP
energy stored in
tRNA-amino acid bond


unstable
so it can release amino acid
at ribosome
Ribosomes

Facilitate coupling of
tRNA anticodon to
mRNA codon


organelle or enzyme?
Structure
ribosomal RNA (rRNA)
& proteins
 2 subunits



large
small
Ribosomes

P site (peptidyl-tRNA site)


A site (aminoacyl-tRNA site)


holds tRNA carrying growing
polypeptide chain
holds tRNA carrying next amino acid to
be added to chain
E site (exit site)

empty tRNA
leaves ribosome
from exit site
Building a Polypeptide

Initiation



brings together mRNA,
ribosome subunits,
proteins & initiator tRNA
Elongation
Termination
Elongation: Growing a Polypeptide
Termination: Release Polypeptide

Release factor
“release protein” bonds to A site
 bonds water molecule to polypeptide chain

Now what happens to the polypeptide?
Protein Targeting

Signal peptide

address label
Destinations:





ex. start of a secretory pathway

secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
RNA polymerase
DNA
Can you tell the
eukaryotic story?
amino
acids
exon
intron
pre-mRNA
tRNA
5' cap
mature mRNA
aminoacyl tRNA
synthetase
polyA tail
large subunit
3'
polypeptide
ribosome
5'
tRNA
small subunit
E P A
Putting it all
together…
Don’t
forget the
YouTube
videos!