School of Medicine

Biochemistry

Protein Synthesis
by

Dr Liks
Protein Synthesis (Outline)
Transcription

Translation

Post-translational Modifications of Proteins

 Genes and Proteins

Only 1.5% of total human DNA is “coding” or exon DNA

 Genes and Proteins

Transcription

Translation
The Genetic Code
 Name the Amino Acids

• GGG?
• UCA?
• CAU?
• GCA?
• AAA?
Degeneracy of the Genetic Code
 Types of RNA
RNAs have a broader range of functions, and several classes are found in cells.
1. Ribosomal RNAs (rRNAs) are components of ribosomes, the complexes that
carry out the synthesis of proteins.

2. Messenger RNAs (mRNAs) are intermediaries, carrying genetic information

from one or a few genes to a ribosome, where the corresponding proteins can
be synthesized.

3. Transfer RNAs (tRNAs) are adapter molecules that faithfully translate the
information in mRNA into a specific sequence of amino acids

4. Small nuclear RNA (snRNA) and microRNA (miRNA) together with proteins
form small nuclear ribonucleoproteins (snRNPS) that act as ribozymes
Transfer RNA (tRNA)
64 Codons and 32 Anticodons
Genetic Information Pathway
Nuclear
DNA membrane

Transcription
Pre-mRNA

RNA Processing
mRNA

Ribosome

Translation

Protein
Transcription: RNA synthesis
 Transcription is a DNA-directed synthesis
of RNA
• Messenger RNA is transcribed from the template strand of a
gene.
• RNA polymerase separates the DNA strands at the appropriate
point and bonds the RNA nucleotides as they base-pair along
the DNA template.
• Like DNA polymerases, RNA polymerases can add nucleotides
only to the 3’ end of the growing polymer.
• Genes are read 3’->5’, creating a 5’->3’ RNA molecule.
Transcription is a DNA-directed synthesis
of RNA
• Specific sequences of nucleotides along the DNA mark where gene
transcription begins and ends.
• RNA polymerase attaches and initiates transcription at the promoter, “upstream” of
the information contained in the gene, the transcription unit.
• The terminator signals mark the end of transcription.
• Bacteria have a single type of RNA polymerase that synthesizes all RNA
molecules.
• In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their
nuclei.
• RNA polymerase II is used for mRNA synthesis.
3 Stages of RNA Synthesis
 Transcription Initiation in Prokaryotes
• The presence of a promoter sequence
determines which strand of the DNA
helix is the template.
• Within the promoter is the starting point
for the transcription of a gene.

• The promoter also includes a binding site

for RNA polymerase several dozen
nucleotides upstream of the start point.

• In prokaryotes, RNA polymerase can

recognize and bind directly to the promoter
region.
 Transcription Initiation in Eukaryotes
• In eukaryotes, proteins called
transcription factors recognize the
promoter region, especially a TATA
box, and bind to the promoter.

• After they have bound to the

promoter, RNA polymerase
binds to transcription factors to
create a transcription
initiation complex.

• RNA polymerase then starts

transcription.
 Elongation of RNA Transcript
• As RNA polymerase moves along
the DNA, it untwists the double
helix, 10 to 20 bases at time.

• The enzyme adds nucleotides to

the 3’ end of the growing strand.

• Behind the point of RNA synthesis,

the double helix re-forms and the
RNA molecule peels away.
 Termination of Transcription
• Transcription proceeds until after the RNA polymerase transcribes a
terminator sequence in the DNA.
• In prokaryotes, RNA polymerase stops transcription right at the
end of the terminator.
• Both the RNA and DNA is then released.

• In eukaryotes, the polymerase continues for hundreds of

nucleotides past the terminator sequence, AAUAAA.
• At a point about 10 to 35 nucleotides past this sequence, the
pre-mRNA is cut from the enzyme.
Eukaryotic cells modify RNA after transcription
• Enzymes in the eukaryotic nucleus modify pre-mRNA before the
genetic messages are dispatched to the cytoplasm.

• At the 5’ end of the pre-mRNA molecule, a modified form of

guanine nucleotide is added, the 5’ cap.
• This helps protect mRNA from hydrolytic enzymes.
• It also functions as an “attach here” signal for ribosomes.
Eukaryotic cells modify RNA after transcription
• At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the
poly(A) tail.
• In addition to inhibiting hydrolysis and facilitating ribosome attachment,
the poly(A) tail also seems to facilitate the export of mRNA from the
nucleus.
• The mRNA molecule also includes non-translated leader and trailer
segments.
 RNA Splicing

• RNA splicing removes introns and joins exons to

create an mRNA molecule with a continuous
coding sequence.
(1) Pre-mRNA combines
with snRNPs and other
proteins to form a
spliceosome.

(2) Within the spliceosome,

snRNA base-pairs with
nucleotides at the ends of
the intron.

(3) The RNA transcript is

cut to release the intron,
and the exons are spliced
together; the spliceosome
then comes apart, releasing
mRNA, which now
contains only exons.
 Roles of RNA Splicing
• RNA splicing appears to have several functions.
• First, at least some introns contain sequences that control gene activity in some
way.
• Splicing itself may regulate the passage of mRNA from the nucleus to the
cytoplasm.
• One clear benefit of split genes is to enable a gene to encode for more than one
polypeptide.

• Alternative RNA splicing gives rise to two or more different polypeptides,

depending on which segments are treated as exons.
• Early results of the Human Genome Project indicate that this phenomenon may
be common in humans.
Protein Synthesis: Translation
70S Ribosomes Vs 80S Ribosomes
Components Required for the Five Major
Stages of Protein Synthesis in E. coli
 Stage 1: Activation of Amino Acids

Aminoacyl-tRNA synthetases attach the correct amino acids to their tRNAs

Stage 2- Formation of The Initiation
Complex in Bacteria
 Initiation: mRNA Shine-Dalgarno
Sequence Interaction with 16S rRNA
Protein Factors Required for Initiation of Translation in Bacteria
and Eukaryotic Cells
Stage 2- Formation of The Initiation
Complex in Eukaryotic Cells
 Stage 3: Elongation
Cells use three steps to add each amino acid residue, and the steps are
repeated as many times as there are residues to be added.

Elongation Step 1: Binding of an incoming aminoacyl-tRNA

Elongation Step 2: Peptide bond formation

Elongation Step 3: Translocation

Elongation Step 1: Binding of an Incoming Aminoacyl-tRNA
Elongation Step 2: Peptide Bond Formation

The peptidyl transferase catalyzing this reaction is the 23S rRNA ribozyme.
 Elongation Step 3: Translocation

The ribosome moves one codon toward the 3’ end of the mRNA, using energy provided by hydrolysis of GTP
bound to EF-G (translocase).
 Elongation Stage of Eukaryotic Protein Synthesis

• The elongation cycle in eukaryotes is quite similar to that in prokaryotes.

• Three eukaryotic elongation factors (eEF1α, eEF1βγ, and eEF2) have

functions analogous to those of the bacterial elongation factors (EF-Tu,
EF-Ts, and EF-G, respectively).

• Eukaryotic ribosomes do not have an E site; uncharged tRNAs are

expelled directly from the P site.
Stage 4: Termination of Polypeptide Synthesis
Requires a Special Signal

Termination of protein synthesis in bacteria. Termination

occurs in response to a termination codon in the A site. First,
a release factor, RF (RF-1 or RF-2, depending on which termination
codon is present), binds to the A site. This leads to hydrolysis of the
ester linkage between the nascent polypeptide and the tRNA in the P
site and release of the completed polypeptide. Finally, the mRNA,
deacylated tRNA, and release factor leave the ribosome, and the
ribosome dissociates into its 30S and 50S subunits.
Stage 5: Post-translational Modifications of Proteins

Newly synthesized polypeptide chains undergo folding and

processing such as:
1. Amino-terminal and carboxyl-terminal modifications
2. Loss of signal sequences
3. Modification of individual amino acids
4. Attachment of carbohydrate side chains
5. Addition of isoprenyl groups
6. Addition of prosthetic groups
7. Proteolytic processing
8. Formation of disulfide cross-links
Post-translational Modifications of Insulin