Download G - AP Bio Take 5

From Gene
to Protein
How Genes
Work
2007-2008
What do genes code for?

How does DNA code for cells & bodies?

DNA
how are cells and bodies made from the
instructions in DNA
proteins
cells
bodies
The “Central Dogma”

Flow of genetic information in a cell

How do we move information from DNA to proteins?
DNA
replication
RNA
protein
DNA gets
all the glory,
but proteins do
all the work!
trait
Metabolism taught us about genes

Inheritance of metabolic diseases
suggested that genes coded for enzymes
 each disease (phenotype) is caused by
non-functional gene product





lack of an enzyme
Tay sachs
PKU (phenylketonuria)
albinism
metabolic pathway
A

enzyme 1
Am I just the
sum of my proteins?
disease
disease
disease
disease
B
C
D
E

enzyme 2

enzyme 3

enzyme 4
a
a
From gene to protein
nucleus
cytoplasm
transcription
DNA
a
a
translation
mRNA
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
Transcription
from
DNA nucleic acid language
to
RNA nucleic acid language
2007-2008
Facts about RNA


ribose sugar
N-bases
uracil instead of thymine
U : A
C : G



single stranded
lots of RNAs

DNA
mRNA, tRNA, rRNA, siRNA…
transcription
RNA
Transcription: Making mRNA
The parts:
 transcribed DNA strand = template strand
 untranscribed DNA strand = coding strand
 The complementary RNA strand is same
sequence as coding strand
 Uses enzyme: RNA polymerase
coding strand
build RNA 535C
(same as DNA G
coding strand) 3
A
G
T
A T C
T A
G
A G C
A
T
C G T
A
C
T
3
G C A U C G U
C
G T A G C A
T
T
A
C
A G
C T
G
A
T
A
T
3
5
unwinding
rewinding
mRNA
5
RNA polymerase
template strand
The Enzymes! RNA polymerases
There are 3 RNA polymerase enzymes

RNA polymerase 1



RNA polymerase 2


transcribes genes into mRNA
RNA polymerase 3


only transcribes rRNA genes which
go to ribosomes
makes ribosomes
only transcribes tRNA genes
each has a specific promoter sequence
it recognizes
How to „know‟ which gene is read? (2
regions)

Promoter region
Located before gene
 Begins with „TATA‟ box binding site
 What does it do? This is the binding site for
RNA polymerase
& transcription
factors


Enhancer region
Additional
binding site far
upstream of gene
What are Transcription Factors?
They are Initiation complex („start‟ complexes)

transcription factors bind to promoter region



This is a suite of proteins which bind to DNA
turn on or off transcription
They trigger the binding of RNA polymerase to DNA
RNA polymerase : Matches bases of
DNA & RNA

Match RNA bases to DNA
bases on one of the DNA
strands
A
G
C
A
G
G
U
U
C
A
AG
U
C
G
A
U
A
C
5'
RNA
A C C polymerase G
A
U
3'
T G G T A C A G C T A G T C A T CG T A C CG T
U
C
Eukaryotic genes have junk!

Eukaryotic genes are not continuous

exons = the real gene


expressed / coding DNA
introns = the junk

introns
come out!
inbetween sequence
intron = noncoding (inbetween) sequence
eukaryotic DNA
exon = coding (expressed) sequence
mRNA splicing – after transcription
What is spliced?
primary transcript = This is pre-mRNA
 When mRNA is spliced….the INTRONS are edited
out!
 make mature (final) mRNA transcript
intron = noncoding (inbetween) sequence
~10,000 base
eukaryotic DNA
exon = coding (expressed) sequence
pre-mRNA
primary mRNA
transcript
mature mRNA
transcript
~1,000 base
spliced mRNA
Splicing must be accurate

No room for mistakes!

a single base added or lost throws off the
reading frame
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGUCCGAUAAGGGCCAU
AUG|CGG|UCC|GAU|AAG|GGC|CAU
Met|Arg|Ser|Asp|Lys|Gly|His
AUGCGGCTATGGGUCCGAUAAGGGCCAU
AUGCGGGUCCGAUAAGGGCCAU
AUG|CGG|GUC|CGA|UAA|GGG|CCA|U
Met|Arg|Val|Arg|STOP|
RNA splicing enzymes

CALLED: snRNPs (small nuclear RNA)


proteins
Spliceosome


snRNPs
snRNA
intron
exon
exon
several snRNPs
5'
recognize splice site sequence

3'
cut & paste gene
spliceosome
5'
3'
lariat
5'
mature mRNA
exon
5'
3'
exon
3'
excised
intron
Alternative splicing

Alternative mRNAs produced from same gene


when is an intron not an intron…
different segments can be treated as exons
Starting to get
hard to
define a gene!
More post-transcriptional processing

Need to protect mRNA on its trip from
nucleus to cytoplasm…WHY?
enzymes in cytoplasm attack mRNA
How does protection occur?

protect the ends of the molecule by:
 add 5 GTP cap
 add poly-A tail
 longer tail, mRNA lasts longer: produces more protein
3'
mRNA
5'
P
G P
P
A
a
a
From gene to protein
nucleus
cytoplasm
transcription
DNA
a
a
translation
mRNA
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
trait
Translation
from
nucleic acid language
to
amino acid language
2007-2008
How does mRNA code for proteins?
DNA
4 ATCG
TACGCACATTTACGTACGCGG
mRNA
AUGCGUGUAAAUGCAUGCGCC
4 AUCG
protein
?
Met Arg Val Asn Ala Cys Ala
20
How can you code for 20 amino acids
with only 4 nucleotide bases (A,U,G,C)?
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNA
AUGCGUGUAAAUGCAUGCGCC
?
protein
Met Arg Val Asn Ala Cys Ala
Who Cracked the code?

1960 | 1968
Nirenberg & Khorana
Crick

determined 3-letter (triplet) codon system
WHYDIDTHEREDBATEATTHEFATRAT

Nirenberg (47) & Khorana (17)
determined mRNA–amino acid match
 added fabricated mRNA to test tube of
ribosomes, tRNA & amino acids



created artificial UUUUU… mRNA
found that UUU coded for phenylalanine
The code

Code for ALL life!


strongest support for
a common origin for
all life
Code is redundant

several codons for
each amino acid

Start codon



AUG
methionine
Stop codons

UGA, UAA, UAG
How are the codons matched to
amino acids?
DNA
3
5
5
3
TACGCACATTTACGTACGCGG
mRNA
AUGCGUGUAAAUGCAUGCGCC
3
codon
5
UAC
tRNA
amino
acid
GCA
anti-codon
CAU
Met
Arg
Val
Same code
as DNA!
a
a
From gene to protein
nucleus
cytoplasm
transcription
DNA
a
a
translation
mRNA
a
a
a
a
a
a
a
a
a
a
a
protein
a
a
a
a
a
a
a
ribosome
aa
trait
Transfer RNA 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
bond requires energy



ATP  AMP
bond is unstable
so it can release amino acid at ribosome easily
Trp C=O
OH
OH
Trp C=O
O
Trp
H2O
O
activating
enzyme
tRNATrp
anticodon
tryptophan attached
to tRNATrp
AC C
UGG
mRNA
tRNATrp binds to UGG
condon of mRNA
Ribosomes

Facilitate coupling of
tRNA anticodon to
mRNA codon
IS this an
organelle or enzyme?


Structure
ribosomal RNA (rRNA) & proteins
 2 subunits



large
small
E P A
Ribosomes

A site (aminoacyl-tRNA site)


P site (peptidyl-tRNA site)


holds tRNA carrying next amino acid to
be added to chain
holds tRNA carrying growing
polypeptide chain
Met
E site (exit site)

empty tRNA
leaves ribosome
from exit site
U A C
A U G
5'
E
P
A
3'
Building a polypeptide

Initiation


Elongation


brings together mRNA, ribosome
subunits, initiator tRNA
adding amino acids based on
codon sequence
Termination

3 2 1
end codon
Leu
Val
Met
Met
Met
Met Leu
Ala
Leu
Leu
release
factor
Ser
Trp
tRNA
U AC
5'
C U GA A U
mRNA A U G
3'
E P A
5'
UAC GAC
A U G C U GA A U
5'
3'
U A C GA C
A U G C U G AAU
5'
3'
U AC G A C
AA U
AU G C UG
3'
A CC
U GG U A A
3'
Protein targeting

See role of Signal peptide
Destinations:




Start
here
inside
cell


start of a secretory pathway

secretion
nucleus
mitochondria
chloroplasts
cell membrane
cytoplasm
etc…
RNA polymerase
DNA
Can you tell
the story?
amino
acids
exon
intron
tRNA
pre-mRNA
5' GTP cap
mature mRNA
aminoacyl tRNA
synthetase
poly-A tail
large ribosomal subunit
polypeptide
5'
small ribosomal subunit
tRNA
E P A
ribosome
3'
The Transcriptional unit (gene?)
enhancer
1000+b
20-30b
3'
RNA
TATA
polymerase
translation
start
TAC
translation
stop
exons
transcriptional unit (gene)
5'
DNA
ACT
DNA
UTR
promoter
UTR
introns
transcription
start
transcription
stop
5'
pre-mRNA
5'
GTP mature mRNA
3'
3'
AAAAAAAA
Bacterial chromosome
Protein
Synthesis in
Prokaryotes
Transcription
mRNA
Psssst…
no nucleus!
Cell
membrane
Cell wall
2007-2008
Prokaryote vs. Eukaryote genes

Prokaryotes

Eukaryotes


DNA in cytoplasm
circular
chromosome
naked DNA

no introns





DNA in nucleus
linear
chromosomes
DNA wound on
histone proteins
introns vs. exons
introns
come out!
intron = noncoding (inbetween) sequence
eukaryotic
DNA
exon = coding (expressed) sequence
Translation in Prokaryotes

Transcription & translation are simultaneous
in bacteria
DNA is in
cytoplasm
 no mRNA
editing
 ribosomes
read mRNA
as it is being
transcribed

Translation: prokaryotes vs. eukaryotes

Differences between prokaryotes &
eukaryotes

time & physical separation between
processes


takes eukaryote ~1 hour
from DNA to protein
no RNA processing
Any Questions??
What color would a smurf turn
if he held his breath?
2007-2008
Substitute Slides
for Student Print version
2007-2008
Can you tell
the story?
The Transcriptional unit
enhancer
exons
1000+b
20-30b
3'
RNA
TATA
polymerase
TAC
transcriptional unit
5'
DNA
ACT
introns
5'
3'
5'
3'