Protein Synthesis Notes
Ch 17
Central Dogma
• DNA
RNA
Protein
Prokaryotic vs Eukaryotic Transcription
Prokaryotic Transcription
-takes place in cytoplasm
-circular DNA w/no histones
-no introns / no mRNA processing
Eukaryotic Transcription
-takes place in nucleus
-DNA has histones and linear
-introns and mRNA processing
Prokaryotic Transcription
STEPS
1. Initiation – RNA polymerase
binds to promoter
2. Elongation – RNA polymerase
reads template 3’-5’ and builds
mRNA strand 5’-3’
3. Termination – mRNA is released …
forms hairpin loop.
Eukaryotic Transcription
1. Initiation – Transcription factors
adhere to the TATA box in
the promoter signaling RNA
Polymerase II to attach. Additional
transcription factors attach and
the transcription initiation
complex is formed. Enhancers/silencers
2.
Elongation – RNA Polymerase II unzips
the DNA and pairs the template with
complementary mRNA nucleotides.
3.
Termination – RNA Polymerase II reaches
the polyadenylation (AAUAAA)
sequence and releases the pre-mRNA.
transcription
*Gene expression is most often regulated at transcription
HHMI transcription initiation complex
Eukaryotic Transcription
Transcription
mRNA processing
mRNA Processing (takes place in the nucleus)
1. Add 5’ cap (guanine with 3 phosphates)
2. Add Poly A tail (repeated Adenines)
3. Introns (non coding regions) cut out
4. Exons (coding regions) fused together
snRNPs (small nuclear ribonucleoproteins)
• snRNPs- small nuclear RNA
– RNA and proteins
• Spliceosome - several snRNPs that
carry out RNA splicing
Spliceosome Animation
Harvard Ribozymes
Ribozyme
Ribozyme – RNA that functions
as an enzyme
-splicing without proteins!
How:
1.
Single stranded and forms 3-D structure
2.
Bases in RNA contain functional groups that may catalyze rxns.
3.
Forms hydrogen bonds
Thomas-Cech Ribozymes
Sidney Altman Thomas Cech
Yale
U of Colorado
Animations
•
•
•
•
•
http://wwwclass.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a2.html
http://www.stolaf.edu/people/giannini/flashanimat/molgenetics/transcription.swf
http://www.concord.org/~btinker/workbench_web/models/eukTranscription.swf
http://vcell.ndsu.edu/animations/transcription/index.htm
http://www.hhmi.org/biointeractive/dna/animations.html
Translation
from
nucleic acid language
to
amino acid language
2007-2008
Translation
• Codons
– 3 nucleotides
decoded into
the sequence of
amino acids
Bacterial chromosome
Translation in
Prokaryotes
Transcription
mRNA
Translation
Psssst…
no nucleus!
protein
Cell
membrane
Cell wall
2007-2008
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
– RNA processing
Translation in Eukaryotes
2007-2008
From gene to protein
transcription
DNA
translation
mRNA
mRNA leaves
nucleus through
nuclear pores
nucleus
a
a
a
a
a
a
a
a
a
a
protein
a
a
ribosome
a
a
a
a
proteins synthesized by
ribosomes using
instructions on mRNA
cytoplasm
How does mRNA code for proteins?
DNA
TACGCACATTTACGTACGCGG
4 ATCG
mRNA
4 AUCG
protein
20
AUGCGUGUAAAUGCAUGCGCC
?
Met Arg Val Asn Ala Cys Ala
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNAAUGCGUGUAAAUGCAUGCGCC
AUGCGUGUAAAUGCAUGCGCC
Met
protein
Cys
Ala
?
Arg Val
Asn
Ala
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 the
wobble good?

Start codon



AUG
methionine
Stop codons

UGA, UAA, UAG
Translation
from
nucleic acid language
to
amino acid language
2007-2008
Translation
• Codons
– blocks of 3
nucleotides
decoded into
the sequence of
amino acids
Bacterial chromosome
Translation in
Prokaryotes
Transcription
mRNA
Translation
Psssst…
no nucleus!
protein
Cell
membrane
Cell wall
2007-2008
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
– RNA processing
Translation in Eukaryotes
2007-2008
From gene to protein
transcription
DNA
translation
mRNA
mRNA leaves
nucleus through
nuclear pores
nucleus
a
a
a
a
a
a
a
a
a
a
protein
a
a
ribosome
a
a
a
a
proteins synthesized by
ribosomes using
instructions on mRNA
cytoplasm
How does mRNA code for proteins?
DNA
TACGCACATTTACGTACGCGG
4 ATCG
mRNA
4 AUCG
protein
20
AUGCGUGUAAAUGCAUGCGCC
?
Met Arg Val Asn Ala Cys Ala
mRNA codes for proteins in triplets
DNA
TACGCACATTTACGTACGCGG
codon
mRNAAUGCGUGUAAAUGCAUGCGCC
AUGCGUGUAAAUGCAUGCGCC
Met
protein
Cys
Ala
?
Arg Val
Asn
Ala
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 the
wobble good?

Start codon



AUG
methionine
Stop codons

UGA, UAA, UAG
How are the codons matched to amino
acids?
DNA
mRNA
3
5
5
3
TACGCACATTTACGTACGCGG
AUGCGUGUAAAUGCAUGCGCC
codon
3
5
tRNA
UAC
amino
acid
Met
GCA
Arg
CAU
Val
anti-codon
From gene to protein
transcription
DNA
translation
mRNA
a
a
a
a
a
a
a
a
a
a
protein
a
a
ribosome
a
a
a
a
aa
nucleus
cytoplasm
Transfer RNA structure
• “Clover leaf” structure
– anticodon on “clover leaf” end
– amino acid attached on 3 end
tRNA – “Wobble”
Inosine can pair with C, A, U allowing for less tRNA’s,
More relaxed at the 3rd base position.
Loading tRNA
• Aminoacyl tRNA synthetase
– enzyme which bonds amino acid to tRNA
– bond requires energy
• ATP  AMP
– energy stored in tRNA-amino acid bond
• 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
• Structure
– ribosomal RNA (rRNA) & proteins
– 2 subunits
• large
• Small
Assembled in the nucleus of Eukaryotes
E P A
Translation Animation McGraw Hill
Uconn Translation Animation
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
U A C
A U G
5'
E
P
A
3'
Building a polypeptide
• Initiation
– brings together mRNA, ribosome
subunits, initiator tRNA
• Elongation
– 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
5'
mRNA
UAC
AA U
A UGC UG
3'
E
P
A
5'
UA C G A C
A UG C U GA AU
5'
3'
U A C GA C
A U G C U G AA U
3'
5'
U A C G A CG A A U
AUG C U
3'
ACC
U GG UA A
3'
Destinations:
Protein targeting

• Signal peptide
– address label





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' cap
mature mRNA
polyA tail
large ribosomal subunit
aminoacyl tRNA
synthetase
3'
polypeptide
5'
small ribosomal subunit
tRNA
E P A
ribosome