Protein Synthesis
Dr. Nalin S Gama-Arachchige
Reference: Campbell NA, Reece JB. 2002. Biology (6th ed). Benjamin Cummings, San Francisco, CA.
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Topic 04: Protein Synthesis
1 hour
Course Objectives: Identify different plant and animal cells/tissues and
explain their functions
Learning Objectives:
Upon completion of this lesson, students will be able to:
•
•
•
Describe the process of protein synthesis
Explain the function of different enzymes in protein synthesis
Given a code, students will be able to ascertain the amino acid
sequence
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www.witnesswell.net
Central Dogma of Molecular Biology
The central dogma of molecular biology explains the flow of
information within a biological system.
DNA
Transcription
RNA
Protein
Translation
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The Flow of Genetic Information
RR/Rr
Rr
Round
Wrinkled
RR/Rr Round
• The information content of DNA is in the form of
specific sequences of nucleotides
• The DNA inherited by an organism leads to specific
traits by dictating the synthesis of proteins
• Proteins are the links between genotype and
phenotype
• Gene expression: the process by which DNA directs
protein synthesis (or, in some cases, just RNAs)
rr Wrinkled
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DNA
molecule
Gene 2
Gene 1
Gene 3
DNA
template
strand
TRANSCRIPTION
mRNA
Codon
TRANSLATION
Protein
Amino acid
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Gene
• A region of specific nucleotide sequence in a chromosome that codes
for a specific polypeptide chain
• The coding sequences (exons) are interrupted by noncoding DNA
(introns)
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Protein Synthesis: Four stages
1. Transcription
2. RNA processing
3. Translation
4. Post-translation processing
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TRANSCRIPTION
The THREE stages of transcription
1. Initiation of Transcription
2. Elongation of the RNA Strand
3. Termination of Transcription
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TRANSCRIPTION
1. Initiation of Transcription
• Promoters signal the initiation of RNA synthesis
• Transcription factors mediate the binding of RNA
polymerase and the initiation of transcription
• The completed assembly of transcription factors and RNA
polymerase II bound to a promoter is called a transcription
initiation complex
• A promoter called a TATA box is crucial in forming the
initiation complex in eukaryotes
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TRANSCRIPTION
Transcription Factors
• Transcription factors are proteins that bind to DNA near the
start of transcription of a Gene.
• Transcription factors either inhibit or assist RNA polymerase in
initiation and maintenance of transcription.
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TRANSCRIPTION
2. Elongation of Transcription
• As RNA polymerase moves along the DNA, it untwists
the double helix, 10 to 20 bases at a time
• Transcription progresses at a rate of 40 nucleotides per
second in eukaryotes
• A gene can be transcribed simultaneously by several
RNA polymerases
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Promoter
Transcription unit
5’
3’
Start point
RNA polymerase
3’
5’
DNA
1
Initiation
5’
3’
3’
5’
RNA
transcript
Unwound
DNA
Template strand
of DNA
2 Elongation
Rewound
DNA
5’
3’
3’
5’
3’
5’
RNA
transcript
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Nontemplate
strand of DNA
Elongation
RNA
polymerase
3’
RNA nucleotides
3’ end
5’
5’
Direction of
transcription
(“downstream”)
Newly made
RNA
Template
strand of DNA
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TRANSCRIPTION
3. Termination of Transcription
• In eukaryotes, the polymerase continues transcription
after the pre-mRNA is cleaved from the growing RNA
chain; the polymerase eventually falls off the DNA
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Promoter
Transcription unit
5’
3’
Start point
RNA polymerase
3’
5’
DNA
1
Initiation
5’
3’
3’
5’
RNA
transcript
Unwound
DNA
Template strand
of DNA
2 Elongation
Rewound
DNA
5’
3’
3’
5’
3’
5’
RNA
transcript
3 Termination
5’
3’
3’
5’
3’
5’
Completed RNA transcript
(Pre-mRNA)
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RNA PROCESSING
• Enzymes in the eukaryotic nucleus modify pre-mRNA before
the genetic messages are dispatched to the cytoplasm
• During RNA processing, both ends of the primary transcript are
usually altered
• Also, usually some interior parts of the molecule are cut out,
and the other parts spliced together
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RNA PROCESSING
• Each end of a pre-mRNA molecule is modified in a particular way:
– The 5 end receives a modified nucleotide 5 cap
– The 3 end gets a poly-A tail
• These modifications share several functions
– They seem to facilitate the export of mRNA
– They protect mRNA from hydrolytic enzymes
– They help ribosomes attach to the 5 end
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RNA PROCESSING
Protein-coding segment
Polyadenylation signal
5’
G
P
P
5’ Cap
3’
P
AAUAAA
5’ UTR
Start codon
A modified guanine nucleotide
added to the 5′ end
Stop codon
3’ UTR
AAA … AAA
Poly-A tail
50–250 adenine nucleotides
added to the 3′ end
RNA processing: addition of the 5 cap and poly-A tail
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RNA PROCESSING
• Most eukaryotic genes and their RNA transcripts have long
noncoding stretches of nucleotides that lie between coding
regions
• These noncoding regions are called intervening sequences,
or introns
• The other regions are called exons because they are
eventually expressed, usually translated into amino acid
sequences
• RNA splicing removes introns and joins exons, creating an
mRNA molecule with a continuous coding sequence
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RNA PROCESSING
5’ Exon Intron
Pre-mRNA
Exon
Exon
Intron
3’
Poly-A tail
5’ Cap
1
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Coding
segment
mRNA
5’ Cap
1
5’ UTR
104
146
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Introns cut out and
exons spliced together
Poly-A tail
146
3’ UTR
RNA processing: RNA splicing
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RNA PROCESSING
• RNA splicing is carried out by
spliceosomes
• Spliceosomes consist of a variety of
proteins and several small nuclear
ribonucleoproteins (snRNPs) that
recognize the splice sites
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5’
RNA transcript (pre-mRNA)
Exon 1
Intron
Exon 2
Protein
snRNA
Other
proteins
snRNPs
Spliceosome
5’
Spliceosome
components
5’
mRNA
Exon 1
Exon 2
Cut-out
intron
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TRANSLATION
Translation is the process
by which ribosomes read
the genetic message in
the mRNA and produce a
protein product according
to the instruction that
message
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Ribosomes
• Ribosomes
facilitate
specific coupling of tRNA
anticodons with mRNA
codons
in
protein
synthesis
• The
two
ribosomal
subunits (large and small)
are made of proteins and
ribosomal RNA (rRNA)
Growing polypeptide
Exit tunnel
tRNA molecules
Large subunit
EP
A
Small subunit
5’
mRNA
3’
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Computer model of functioning ribosome
Ribosomes
P site
(Peptidyl-tRNA binding site)
A site
(Aminoacyl-tRNA binding site)
tRNA
The ribosome has multiple
binding sites:
– P site – binds the tRNA
attached to the growing
peptide chain
E P
– A site – binds the tRNA
carrying the next amino acid
– E site – binds the tRNA that
carried the last amino acid
mRNA binding site
Large subunit
A
Large subunit
E site (Exit site)
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Schematic model showing binding sites
Ribosomes
The ribosome has two primary functions
– decode the mRNA
– form peptide bonds
Peptidyl transferase is the enzymatic component of the
ribosome which forms peptide bonds between amino acids
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tRNA
tRNA molecules carry amino acids to the ribosome for
incorporation into a polypeptide
– aminoacyl-tRNA synthetases add amino acids to the amino
acid attachment site of tRNA
– the anticodon loop contains 3 nucleotides complementary
to mRNA codons
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3’
Amino acid
attachment site
PO4
5’
Amino acid
attachment site
5’
3’
T loop
Hydrogen
bonds
D loop
Hydrogen
bonds
Variable loop
5’
3’
Anticodon
Anticodon
Anticodon
Two-dimensional structure of tRNA
Three-dimensional structure of tRNA
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Twenty
Amino
Acids
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The codon table for31mRNA
Growing polypeptide chain
Next amino acid to be added
to polypeptide chain
Amino end
Large subunit
tRNA
E
mRNA
5’
Codons
3’
Small subunit
Schematic model with mRNA and tRNA
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TRANSLATION
The THREE stages of translation
1. Initiation of Translation
2. Elongation of the Polypeptide Chain
3. Termination of Translation
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TRANSLATION
1. Initiation of Translation
• The initiation stage of translation brings together mRNA, a
tRNA with the first amino acid, and the two ribosomal
subunits
• First, a small ribosomal subunit binds with mRNA and a special
initiator tRNA
• Then the small subunit moves along the mRNA until it reaches
the start codon (AUG)
• Proteins called initiation factors bring in the large subunit that
completes the translation initiation complex
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Large ribosomal subunit
3’ U A C 5’
5’ A U G 3’
Initiator
tRNA
P site
GTP
GDP
E
mRNA
5’
Start codon
mRNA binding site
3’
Small
ribosomal
subunit
5’
A
3’
Translation initiation complex
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TRANSLATION
2. Elongation of Translation
• During the elongation stage, amino acids are added one by one
to the preceding amino acid
• Each addition involves proteins called elongation factors and
occurs in three steps: codon recognition, peptide bond
formation, and translocation
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Amino end
of polypeptide
E
3’
mRNA
Ribosome ready for
next aminoacyl tRNA
P
A
site site
5’
GTP
GDP
E
E
P A
P A
GDP
GTP
E
P A
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Amino
acids
Polypeptide
Ribosome
tRNA with
amino acid
attached
tRNA
Anticodon
Codons
5’
mRNA
3’
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TRANSLATION
3. Termination of Translation
• Termination occurs when a stop codon in the mRNA reaches
the A site of the ribosome
• The A site accepts a protein called a release factor
• The release factor causes the addition of a water molecule
instead of an amino acid
• This reaction releases the polypeptide, and the translation
assembly then comes apart
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Release
factor
Free
polypeptide
5’
3’
5’
5’
Stop codon
(UAG, UAA, or UGA)
3’
2 GTP
3’
2 GDP
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TRANSLATION
Polyribosomes
• A number of ribosomes can translate a single mRNA
simultaneously, forming a polyribosome (or polysome)
• Polyribosomes enable a cell to make many copies of a
polypeptide very quickly
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TRANSLATION
Polyribosomes
Growing
polypeptides
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5’ end)
End of
mRNA
(3’ end)
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POST TRANSLATION PROCESSING
• Often translation is not sufficient to make a functional protein
• Polypeptide chains are modified after translation
• Completed proteins are targeted to specific sites in the cell
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POST TRANSLATION PROCESSING
• During and after synthesis, a polypeptide chain
spontaneously coils and folds into its three-dimensional
shape
• Proteins may also require post-translational modifications
before doing their job
• Some polypeptides are activated by enzymes that cleave
them
• Other polypeptides come together to form the subunits of a
protein
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Targeting Polypeptides to Specific Locations
• Free ribosomes (in the cytosol) mostly synthesize
proteins that function in the cytosol
• Bound ribosomes (attached to the ER) make proteins of
the endomembrane system and proteins that are
secreted from the cell
• Ribosomes are identical and can switch from free to
bound
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Targeting Polypeptides to Specific Locations
• Polypeptide synthesis always begins in the cytosol
• Synthesis finishes in the cytosol unless the polypeptide
signals the ribosome to attach to the ER
• Polypeptides destined for the ER or for secretion are marked
by a signal peptide
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Ribosome
mRNA
Signal
peptide
Signal
peptide
removed
Signalrecognition
particle (SRP)
CYTOSOL
ER LUMEN
SRP
receptor
protein
ER
membrane
Protein
Translocation
complex
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RR/Rr Round
rr Wrinkled
The Flow of Genetic Information
The pea plant can produce smooth or wrinkled seeds.
Wrinkled peas taste sweet.
• When functioning normally, the
SBE1(starch-branching enzyme 1)
enzyme builds starch.
• A mutation took placed in the coding
sequence of the SBE1 gene. disables the
protein.
• The job of SBE1 is to add branches to
the chains of sugar.
• The peas can't make branched starch, and
sugar accumulates.
• Branches help starch grow very large,
and break down quickly when the
plant needs energy.
• The sugar attracts water, causing the peas to
swell and then wrinkle as they mature and
dry.
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KEY TERMS: Genes to Proteins
• Trascription
• mRNA
• Translation
• RNA Processing
• TATA box
• Pre-mRNA
• Template Strand
• Codon
• RNA polymerase
• Transcription factors
• Terminator
• 5’ cap
• Poly (A) tail
• RNA splicing
• Intron
• Exon
• Splicesome
• tRNA
• Anti codon
• P A E sites
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