Protein Synthesis:
From Gene to Protein
Step #1 DNA unwinds and exposes the bases (Initiation)
Begins at the Promoter Site
How is a gene found from 3 billion base pairs?
1. TATA Box (25 bases upstream)
a. Area that signals the RNA Polymerase II to attach to the DNA
b. Transcription Factors bind to the TATA Box to help the RNA
Polymerase II to attach to the DNA
2. Transcription Initiation Complex: Transcription Factors + RNA
Polymerase at the promoter site
Promoter Site
TATAAAA
ATATTTT
Template DNA (Gene)
TATA BOX
Initial Transcription Factor
Transcription Factors
Transcription
Initiation
Complex
Why is this important?
RNA Polymerase II
Step #2
mRNA made from DNA (Transcription)
Promoter region
Transcription unit (gene)
Initiation: Covered in Step One
RNA Polymerase
Coding strand
Elongation: RNA Polymerase
moves downstream making RNA
in the 5’  3’ direction of the
template strand
mRNA
Termination Sequence
Termination: RNA
Polymerase released
when a terminator
sequence is reached
mRNA
(AATAAA)
RNA Polymerase
Step #3 mRNA Leaves Nucleus and Goes to the Ribosome
A. RNA Processing:
mRNA is modified before it leaves the nucleus
Pre-mRNA
mRNA used in Translation
Processing
1. Alteration at the ends of the pre-mRNA
Leader
Coding Segment
Start Codon
Trailer
Stop Codon
Termination
Sequence
Addition of the 5’ Cap
Addition of the Poly (A) tail
1. Prevents degradation by enzymes
1. Prevents degradation by enzymes
2. Allows attachment to ribosome
2. Facilitates export from the nucleus
2. Alteration of the coding sequence - mRNA Splicing
Pre-mRNA
5’ Cap Exon
Intron
Exon
mRNA
Exon
Coding Segment
Poly (A) tail
Part of the RNA that remains to be translated into protein
Intron Part of the RNA that is removed
Intron
5’ Cap
Poly (A) tail
Enzymes
5’ Cap
Exon
Intron
Exon 1 + Exon 2 + Exon 3
Coding Segment
Poly (A) tail
RNA Processing Vocabulary
Pre-RNA
Exon
Intron
Exon
Proteins
snRNA
snRNA’s
Small Nuclear RNA
snRNP’s
‘SNURPS” Small
Ribonucleoproteins
Proteins
snRNP’s
Spliceosome
Spliceosome
Ribozymes
The combination of snRNA +
snRNP + Protein used to cut
out introns
RNA that function as
enzymes. Some RNA’s can
splice themselves
What are the Functions of introns and exons?
Spliceosome
Components
Removed Intron
mRNA
1. Allows one code to make
different proteins from generic
“mix and match” domains
2. Controls gene expression
B. Attachment to the Ribosome
A Site
1. Ribosome Anatomy
P site
Large Subunit
E Site
Small Subunit
2. Steps of Attachment (Initiation)
mRNA
binding site
a) mRNA attaches to the smaller unit
b) Initiator tRNA with anticodon UAC bonds to start codon AUG
c) Large ribosomal unit joins small unit with the tRNA at the P site.
d) GTP provides the energy
Met
UA C
A U G
Met
GTP
Slide 9
Step #4 tRNA Bonds to Appropriate Amino Acid
Specific Amino Acid
Requirements
Aminoacyl-tRNA Synthase
1. Aminoacyl-tRNA Synthase enzyme
2. Specific Amino Acid
Active site fits
specific Amino Acid
3. ATP
4. Specific tRNA
tRNA
Active Site specific for anticodon
Aminoacyl tRNA
“activated amino acid”
Step # 5 tRNA bonds to mRNA at rRNA
Step # 6 Amino Acids Bond to make the Protein
All part of
Translation
Steps in Translation
1. Initiation – attachment of mRNA to the ribosome
(This was already covered in Step # 3)
2. Elongation – the addition of amino acids to the growing protein chain
A Site
P site
a)
Codon Recognition : tRNA binds to
codon at A site
E Site
GTP
GDP
GDP
c)
Translocation:
tRNA in P site is
shifted to the E
site
b) Peptide Bond Formation: Amino
acids bond to form protein chain
GTP
Note: Growing Chain
3. Termination: Completed Protein and mRNA released from the Ribosome
Release Factor Protein
a) Release factor protein binds at stop
codon at the A site
Stop Codons
(UAG, UAA, UGA)
b) The release factor breaks
bond of the tRNA and the last
amino acid of the ribosome
releasing the tRNA
c) The 2 ribosomal units, mRNA,
release factor dissociate
Step #7
tRNA goes back to pick up more amino acids;
mRNA breaks down;
Funtional protein ready for use
A. mRNA
1. May not immediately break down but bond to another ribosome to
form a polyribosome
2. Many proteins can be made with one mRNA transcript
B. Protein
Most proteins must be modified to be functional
-amino acids cleaved (methionine), functional groups added, tertiary and quaternary structures
modified
1. Chaperonins needed to fold protein into correct tertiary structure
2. Secretory proteins are targeted to be modified in the Rough ER prior to export
Reading the Code
3’ – TACGGCCGATTCTGACATCGAACT-5’
5’ – ATGCCGGCTAAGACTGTAGCTTGA-3’
3’ – TAC GGC CGA TTC TGA CAT CGA ACT – 5’
5’ - ATG CCG GCT AAG ACT GTA GCT TGA – 3’
mRNA – AUG CCG GCU AAG ACU GUA GCU UGA
(codons)
tRNA – UAC GGC CGA UUC UGA CAU CGA ACU
(anticodons)
Amino Acids = Met – Pro – Ala – Lys – Thr – Val – Ala
Mutations: Changes in the DNA Sequence
Type of Mutation
A. Chromosomal rearrangement
(aneuploidy, trisomy, polyploidy)
B. Changes in DNA base pairs
(Point Mutations)
Typical Effect of Mutation
A. Usually lethal due to high
number of genes (proteins)
involved
B. Effect Varies
1. Silent – no effect due to the
redundancy of the triplet code
2. Missense – Little effect resulting
with a functional protein
3. Nonsense – Protein nonfunctional
C. Point Mutations Types
1. Base- Pair Substitutions
Replacing one base pair with another
2. Deletions – removal of one base pair
3. Insertions – addition of one base pair
Usually silent due to redundancy and
“wobble” of code. Effect depends on:
1)
Amino Acid type changed
2)
Change in start or stop codons
Cause frame shift mutations
forming nonsense proteins
THE END
Slide 6
Slide 3
Slide 8
Ribosome Anatomy
Growing Protein Chain
Large
Subunit
Computer Model
From X-Ray
Crystallography
Data
E
P
A
Small
Subunit
mRNA
Slide 7
A Point Mutation with Bad Effects - Sickle Cell Anemia
Difference in
electronegativity
of this amino
acid causes
hemoglobin to
change shape
after it gives up
it’s O2
No Change due to
redundancy of the
code or “wobble”
Different Amino Acid
Effect depends on the
amino acid type
1. Hydrophobic or
hydrophlic
2. Positive or negative
3. Acidic or basic
A premature
stop codon will
prevent from
forming the
complete protein
THEREDDOGANDCAT
Reading frame
THE RED DOG AND CAT
R
Deletion
frameshift
THE EDD OGA NDC AT
R
Insertion
frameshift
THE RRE DDO GAN DCA T
Genetic Code
Redundancy
1. There are multiple codes
for the same amino acids
2. The most variation is
found in the 3rd base.
“Wobble”
3. Some base pairs will
bond to its “wrong” pair
in the 3rd base
4. Some tRNA use the base
Inosine(I) as their 3rd
base that can bond to
any base
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