TRANSLATION
At the end of this lecture, students should be able to
1. describe the steps of translation
2. describe translocation
3. describe the role of release factors in termination
4. Describe the structure of ribosome and the EPA site with their roles in
synthesis
5. Describe the translation complex
6. describe the role of aminoacyl synthetase and the amount of ATP utilized
7. Describe role of tRNA, mRNA and rRNA in protein synthesis
8. list antibiotics that interfer with protein synthesis and describe their
mechanism
9. List steps involved in post translational modification
10. Describe the steps of collagen synthesis and location in which it occurs.
11. clinical correlate: scruvy, menke’s disease, Ehler’s Dahlors, Osteogenesis
imperfecta, Inclusion cell bogy
12. Describe the role of mannose-6-phosphorylation in ensuring trafficking of
proteins.
13. Describe the role of chaperones in protein processing.
Protein synthesis is complex process in
which genetic information encoded in the
nucleic acids is "translated" into the 20 amino
acid "alphabet" of polypeptides.
In addition to translation, protein
synthesis can also be considered to include the
processes of posttranslational modification and
targeting.
Posttranslational modification consists of
a wide variety of chemical alterations that
cells use to prepare polypeptides for their
functional roles.
Protein synthesis takes place on ribosomes which is a
nucleoprotein and contains 65 percent r-RNA and 35 percent
proteins.
Mg++ is required to hold the two subunits together.
The two subunits in prokaryotes are 50S large subunit
and 30S small subunit while in eukaryotes they are 60S and
40S.
Most of the ribosomal proteins are low molecular weight
basic proteins. Due to their basic charge they can easily interact
with RNA which is negatively charged.
The mitochondrial ribosomes are similar to those of
prokaryotes.
A polysome or polyribosome is a beaded string-like
linear cluster of 5-8 ribosomes on an m-RNA.
Each ribosome has peptidyl (P) and aminoacyl (A)
The process of protein synthesis (after
transcription has taken place) can be divided in
following steps:
1. Activation of amino acids,
2. Initiation,
3. Elongation, and
4. Termination.
Recognition of amino acids
The attachment of amino acids to tRNAs, a process that
is considered to be the first step in protein synthesis, is
catalyzed by a group of enzymes called the aminoacyl-tRNA
synthetases.
In most organisms there is at least one aminoacyl-tRNA
synthetase for each of the 20 amino acids.
The process in which an amino acid is linked to the 3'
terminus of the correct tRNA consists of two sequential
reactions, both of which occur within the active site of the
synthetase.
The sum of the reactions catalyzed by
the aminoacyl-tRNA synthetases is as
follows:
Amino acid + ATP + tRNA
aminoacyl-tRNA + AMP +PP
Because the product PP is
immediately hydrolyzed with a large loss
of free energy, tRNA charging is an
irreversible process.
Translation
Boumphreyfr /Wikipedia
Ribosomes
• Prokaryotes
• 70S ribosomes
• Small (30S) and large (50S) subunit
• Small subunit: 16S RNA plus proteins
• Large subunit: 5S RNA, 23S RNA, plus proteins
• Protein synthesis inhibitor antibiotics
• Aminoglycosides, others
• Target components of bacterial ribosomes
Ribosomes
• Eukaryotes
• 80S ribosomes
• Small (40S) and large (60S) subunits
• Small subunit: 18S RNA plus proteins
• Large subunit: 5S RNA, 28S RNA, 5.8S RNA plus proteins
tRNA
• Transfers amino acids to protein chains
• Synthesized by RNA polymerase III
• Many bases are chemically modified
N,N dimethyl Guanosine Guanosine
tRNA
• Cloverleaf shape (secondary structure)
• Base pairing within molecule
• 70-90 nucleotides in length (tiny)
• Key portions
• Anticodon
• D loop (part of D arm)
• T loop (part of T arm)
• 3’ end
Yikrazuul
Anticodon
• 3 nucleotides on tRNA
• Pairs with complementary mRNA
• Correct pairing →correct protein
synthesis
Boumphreyfr /Wikipedia
Genetic Code
D loop
• Contains dihydrouridine
• tRNA recognition by aminoacyl-tRNA synthetase
Dihydrouridine
Uridine
T loop
• Contains a TΨC sequence
• T = Ribotymidine
• Ψ = Pseudouridine
• C = Cytidine
• Needed for tRNA ribosome binding
Ribothymidine Uridine Pseudouridine
3’ End
• Always ends in CCA
• Hydroyxl (OH) of A attaches to amino acid
Yikrazuul
Charging
• Process of linking amino acids to tRNA
• Each tRNA linked to one amino acid
• Catalyzed by Aminoacyl-tRNA synthetase
• Adds amino acid to tRNA
• Requires ATP
tRNA Aminoacyl
Group
Charged
tRNA
ATP
Amino Acid
AMP
AMP
Aminoacyl-tRNA synthetase
• One enzyme per amino acid in most eukaryotic cells
• i.e. one enzyme attaches glycine to correct tRNA
Dancojocari/Wikipedia
tRNA
• Many amino acids have similar structures
• Mischarged tRNA →wrong AA for mRNA
codon
• Hydrolytic editing
• Aminoacyl-tRNA synthetase scrutinizes amino acid
• If incorrect →hydrolyzes from AMP or tRNA
• Increases accuracy of charging tRNA
Initiation
Translation begins with the formation of an initiation
complex.
mRNA binds to the 30S subunit, it is guided into a precise
location, so that the initiation codon AUG is correctly
positioned.
In the next step in initiation,- the binding of the
initiating tRNA to the initiation codon in the P site. (There are
two sites on the complete ribosome for codone-anticodone
interaction: the P (peptidyl) site and the A (acyl) site. The
initiating tRNA in prokaryotes is N-formyl-methionine-tRNA.
1. Eukaryotic initiation factors (eIFs) identify
the 5′ cap.
2. eIFs help assemble the 40S ribosomal
subunit with the initiator tRNA.
3. eIFs released when the mRNA and the
ribosomal 60S subunit assemble with the complex.
Requires GTP.
Protein Synthesis
• Ribosomes: Four binding sites
• One for mRNA
• Three for tRNA: A-site, P-site, E-site
5’ A 3’
Protein Synthesis
• A-site: Amino acid binding (charged tRNA)
• P-site: tRNA attached to growing protein chain
• E-site: Exit of tRNA
5’ A 3’
Initiation
• Begins with AUG on mRNA
• Codes for methionine or N-formylmethionine (fMet)
• Binds directly to P-site
• Usually removed later by protease enzymes
• fMET = chemotaxis of neutrophils (innate immunity)
Methionine N-formylmethionine
Initiation
• Uses GTP hydrolysis
• In eukaryotes require initiation factors (proteins)
• Assemble ribosomes and tRNA
Elongation
It is during the elongation phase that the polypeptide is actually
synthesized according to the specifications of the genetic message.
Elongation, the phase in which amino acids are incorporated into a
polypeptide chain, consists of three steps: (1) positioning of an
aminoacyl-tRNA in the A site, (2) peptide bond formation, and (3)
translocation.
The prokaryotic elongation process begins when an aminoacyl-
tRNA, specified by the next codon, binds to the A site.
After the positioning of the second aminoacyl-tRNA in the A site,
the formation of a peptide bond is catalyzed by peptidyl transferase.
Uncharged tRNA occupying the P site leaves the ribosome.
For translation to continue, the mRNA must move, or
"translocate," so that a new codon-anticodon interaction can occur. The
unoccupied A site then binds an appropriate aminoacyl-tRNA to the new
A site codon. Elongation continues until a stop codon enters the A site.
Elongation
• Usually divided into a sequence of four steps
• Uses elongation factors (proteins)
• Bacteria: EF-Tu and EF-G
• Eukaryotes: EF1 and EF2
• Hydrolyze GTP to GDP
• EF2: Target of bacterial toxins
• Diphtheria toxin (Corynebacterium diphtheriae)
• Exotoxin A (Pseudomonas aeruginosa)
• Inhibits protein synthesis
Protein Synthesis
• Step 1: Charged tRNA binds A-site
• P-site and A-site next to one another
NH2
t t
5’ 3’
E P A
Protein Synthesis
• Step 2: Amino acid joined to peptide chain
• Catalyzed by ribosome (“ribozyme”)
• Peptidyl transferase: Part of large ribosome (made of RNA)
• Protein attached to A-site
NH2
t t
5’ E P A 3’
Protein Synthesis
• Step 3: Ribosome moves down mRNA toward 3’ end
• “Translocation”
• Protein moves to P-site
NH2
t t
5’ E P A 3’
Protein
Synthesis
• Step 4: tRNA leaves E-site
NH2
t
t
5’ E P A 3’
Termination
• Translation ends at mRNA stop codons
• UAA, UAG, UGA
• Not recognized by tRNA
• Do not specific an amino acid
• Releasing factors bind to ribosome at stop codons
• Catalyze water added to protein chain
NH2
OH
Termination
The termination phase begins when a termination codon
(UAA, UAG, or UGA) enters the A site.
Releasing factors (RF-1 and RF-2) are involved in
termination. The codons UAA and UAG are recognized by RF-
1, whereas UAA and UGA are recognized by RF-2. The
peptidyl transferase hydrolyzes the bond linking the completed
polypeptide chain and the P site tRNA.
Following the polypeptide's release from the ribosome,
the mRNA and tRNA also dissociate. Termination ends with
the dissociation of the ribosome into its constituent subunits.
MOLECULAR CHAPERONES
Proteins translated on the RER generally fold and
assemble into subunits in the ER before being
transferred to the Golgi. Other proteins fold in the
cytoplasm.
Molecular chaperones (proteins such as Calnexin
and BiP) assist in this process of protein folding.
Failure to fold correctly usually results in eventual
destruction of the protein
Proteins that are misfolded are targeted for
destruction. The defective copies are covelently
marked for destruction by the addition of multiple
copies of Ubiquitin. Poly ubiquinated protein are
directed to proteasome for destruction. Proteasome
are large cytoplasmic complexes that have
multiple protease activities capable of digesting
damaged protein to peptides.
In certain genetic diseases, mutation may
cause copies of protein fold incorrectly and
result in loss of protein function and in some
cases accumulation of misfolded protein in
the ER.
Alpha 1 antitrypsin deficiency.- mutation
causes the alpha1 antitrypsin protein to
misfold and aggregate in the endoplasmic
reticulam where it damages cells leading to
cirrhosis. Alpha 1 antirypsin is synthesized by
liver and secreted into blood stream. It
protect cells by serving as inhibitor of
proteases released during a normal
inflamatory response
The majority of cases of cystic fibrosis result
from deletion of phenylalanine at
position(508) which interferes with proper
protein folding and posttranslational
processing of oligosacharide side chains,
abnormal chloride channel protein that
reaches cell membrane contribute to the
pathogenesis of cystic fibrosis
Many proteins require signals to ensure delivery to the appropriate
organelles. Important among these signals are:
The N-terminal hydrophobic signal sequence used to ensure
translation on the RER.
Phosphorylation of mannose residues important for directing an
enzyme to a lysosome.
N-terminal hydrophobic signal sequence:- This sequence is
found on proteins destined to be secreted (insulin), placed in
the cell membrane (Na/K ATPase), or ultimately directed to
the lysosome (sphingomyelinase). These proteins all require
N-terminal hydrophobic signal sequences as part of their
primary structure. Translation begins on free cytoplasmic
ribosomes, but after translation of the signal sequence, the
ribosome is positioned on the ER with the help of signal
recognition particle . The signal sequence is cleaved off in the
ER by the signal peptidase, (translation continues) and then
the protein passes into the Golgi for further modification and
sorting. In transit through ER, Golgi, the protein acqurie
oligosaccharide side chain.
Lysosomal enzymes and phosphorylation of mannose:-
Lysosomal enzymes are glycosylated and modified in a
characteristic way.
Most importantly, when they arrive in the Golgi, specific
mannose residues in their oligosaccharide chains are
phosphorylated.
This phosphorylation is the critical event that removes
them from the secretion pathway and directs them to
lysosomes.
Genetic defects affecting this phosphorylation produces
I-cell disease (coarse facial feature, macroglosia, gingival
hyperplasia, psycho motor, growth retardation etc) in
which lysosome enzymes are released into the
extracellular space and inclusion bodies accumulate in
cells compromising its function.
In Tay-Sachs disease lysosomal enzyme is missing, the
undigested substrate accumulate in the cell often leading to
serious consequences.
.
Tetracyclines are a class of antibiotics that bind to the 30S
ribosomal subunit, thereby blocking the access of the aminoacyl-tRNA
to the mRNA–ribosomal complex. Aminoglycosides, such as
gentamicin, are a class of antibiotics that bind to the 30S ribosomal
subunit, thus interfering with the assembly of the functional ribosomal
apparatus. Both erythromycin and clindamycin are antibiotics that inhibit
protein synthesis as they bind the 50S ribosomal subunit of bacteria. This
results in inhibition of translocation of the growing peptide.
The antibiotic puromycin is an analog of aminoacyl-tRNA. It inhibits
both prokaryotic as well as eukaryotic translation as it acts as a chain
terminator in protein synthesis. Chloramphenicol, an antibiotic
rarely used because of the potential to develop decreased white blood
cells, inhibits peptidyltransferase, thus halting protein synthesis.
Diphtheria toxin is produced from phage genes incorporated into
the bacterium Corynebacterium diphtheriae. The toxin causes
diphtheria, a lethal disease of the respiratory tract. The A fragment of the
toxin catalyzes the adenosine diphosphate (ADP)-ribosylation of EF-2,
thus inhibiting translocation in eukaryotes.
Post-Translation modification:
Primary-sequence of amino acids specified in the gene.
Secondary-folding of the amino acid chain into an
energetically stable structure. Two common examples are the
(X-helix and the pleated sheet). These shapes are reinforced by
hydrogen bonds.
Tertiary-positioning of the secondary structures in relation to
each other to generate higher-order three-dimensional shapes.
Tertiary, structure also includes the shape of the protein as a
whole (globular, fibrous).
Quaternary-in proteins such as hemoglobin that have multiple
subunits, quaternary structure describes the interactions among
subunits.
1. Proteolytic cleavage. Typical examples of proteolytic
cleavage include the removal of the N-terminal methionine residue,
signal sequences, and the conversion of inactive precursors to their
active counterparts. The proteolytic processing of insulin provides a
well-researched example of the conversion of a nonenzyme protein
into its active form.
2. Glycosylation. Although a wide variety of eukaryotic pro
teins are glycosylated, the functional purpose of the carbohydrate
moieties is not always obvious. In general, secreted proteins contain
complex oligosaccharide species, while ER membrane proteins possess
high mannose species.
3. Hydroxylation. Hydroxylation of the amino acids proline and
lysine is required for the structural integrity of the connective tissue
proteins collagen and elastin. Additionally, 4-hydroxyproline is also
found in acetylcholinesterase and complement. Ascorbic acid (vitamin
C) is required for the hydroxylation of proline and lysine residues in
collagen.
4. Phosphorylation. The roles of protein phosphorylation in various examples of
metabolic control and signal transduction are well known. Protein phosphorylation
may also play a critical (and interrelated) role in protein-protein interactions. For
example, the autophosphorylation of tyrosine residues in PDGF receptors ap
parently results in the subsequent binding of certain cytoplasmic signaling
molecules.
5. Lipophilic modifications. The covalent attachment of lipid moieties to
proteins improves membrane binding capacity and/or certain protein-protein
interactions. Among the most common lipophilic modifications is acylation (the
attachment of fatty acids). Although the fatty acid myristate (14:0) is relatively
rare in eukaryotic cells, myristoylation is one of the most common forms of
acylation.
6. Methylation. Protein methylation serves several purposes in eukaryotes.
The methylation of altered aspartate residues by a specific type of
methyltransferase promotes either the repair or the degradation of damaged
proteins. Other methyltransferases catalyze reactions that alter the cellular roles of
certain proteins.
7. Disulfide bond formation. Disulfide bonds are generally found only in
secretory proteins (e.g., insulin) and certain membrane proteins. Cytoplasmic
proteins generally do not possess disulfide bonds because of the presence of
POSTTRANSLATIONAL MODIFICATIONS
OF COLLAGEN
Collagen is an example of a protein that undergoes
several important Co and posttranslational modifications.
It has a somewhat unique primary structure in that much
of its length is composed of a repeating tripeptide Gly-X-
Y-Gly-X-Y- etc, Hydroxyproline is an AA unique to
collagen. Hydroxy prolein is produced by hydroxilation of
prolyl residue at Y position in procollagen chain as they
pass through RER.
Formation of Hydroxyproline & Hydrolysine
The steps in the formation of a collagen fibril
include:
1. Prepro-α-chains containing a hydrophobic signal
sequence synthesized by ribosome attached to RER.
2. The hydrophobic signal sequence is removed by
signal peptidase in the RER to form pro- α-chains.
3. Selected proline and lysine residues are
hydroxylated by prolyl hydroxylase and lysyl
hydroxylase respectively which are located in the
RER, require Vit. C, deficiency of which produces
scurvy.
4. Selected hydroxylysines are glycosylated.
5. Three pro-α-chains assemble to form a triple
helical structure (procollagen), which can now be
transferred to Golgi. Modification of
oligosaccharide continues in the Golgi.
7. Procollagen is secreted from the cell.
8. The propeptides are cleaved from the ends of
procollagen by proteases to form collagen
molecules (also called Tropocollagen).
9. Collagen molecules assemble into fibrils.
Cross-linking involves lysyl oxidase, an enzyme
that requires O2 and Copper.
10. Fibrils aggregate and cross-link to form
collagen fibers. This cross-linking provides
tensile strength necessary for proper
functioning of connective tissue.
Biosynthesis of Collagen
TYPES OF COLLAGEN
Type Tissue distribution
I Skin, bone, tendon
II Cartilage
III Blood vessels
IV Basement membrane
DISORDERS OF COLLAGEN BIOSYNTHESIS
DISEASE DEFECT MAJOR SYMPTOMS
Deficient Petechiae,
Scurvy
hydroxylation ecchymoses, loose
secondary to Vit. teeth, bleeding
C deficiency gums, poor wound
healing.
Mutations in the Fractures, blue
Osteogenesis
gene for type I sclera
imperfecta
collagen
Mutations in Hyperextensible
Ehler-Danlos skin,
type I and III
syndromes hypermobile
collagen gene
and lysine joints, varicose
hydroxylase veins, arterial
gene ruptures,
Signs of vitamin C deficiency
Symptoms can appear within 30days
Golgi is the distribution center for proteins and lipids from the ER to the
vesicles and plasma membrane. Modifies N-oligosaccharides on asparagine.
Adds O-oligosaccharides on serine and threonine. Adds mannose-6-
phosphate to proteins for trafficking to lysosomes.
Endosomes are sorting centers for material from outside the cell or from the
Golgi, sending it to lysosomes for destruction or back to the
membrane/Golgi for further use.
I-cell disease (inclusion cell disease)—inherited lysosomal
storage disorder; defect in N-acetylglucosaminyl-1-phosphotransferase
failure of the Golgi to phosphorylate mannose residues (i.e., mannose-6-
phosphate) on glycoproteins proteins are secreted extracellularly rather
than delivered to lysosomes.
Results in coarse facial features, clouded corneas, restricted joint
movement, and high plasma levels of lysosomal enzymes. Often fatal in
childhood.
Acts as a cofactor for hydroxylation reactions
Collagen synthesis
Collagen has 3 interwoven chains of
glycine, proline and hydroxyproline
Hydroxyproline is crucial as the H-
bonds of its OH groups give chains
stability
Hydroxylysine also required for x-links
Formation of hydroxyproline and
hydroxylysine require vitamin C as a
cofactor
Proline hydroxylase Lysine hydroxylase
Proline hydroxyproline Lysine hydroxylysine
Vit C + Vit C +
Fe2+ Fe2+
Scurvy
Menkes disease:- X-linked recessive condition,
caused by the mutations in the genes encoding Cu
efflux protein. Absorbed Cu gets trapped in the
intestinal epithelial cells as the Cu can’t be released
from the cell and there is functional Cu deficiency.
This results in deficient cross linking. Cross linking
involves lysyl oxydase ( it needs 02 and copper)
Symptoms include Depigmented (steely) hair,
arterial tortuosity, osteoporosis, cerebral
degeneration etc.
Ehlers-Danlos syndrome and Osteogenesis
imperfecta are the examples for locus
heterogeneity.
The bacterial ribosome and its involvement in protein
synthesis pathways hold great importance to the
function of various antibiotics that antagonize various
steps in the process. Which of the following is true of the
50S ribosomal subunit in bacterial protein synthesis?
A) Proteins in the 50S subunit are responsible for the
creation of a peptide bond
B) Streptomycin binds to the 50S subunit, disrupting the
translocation step
C) The 23S rRNA molecule within the 50S subunit is
responsible for the creation of a peptide bond
D) The 50S subunit holds the binding site for both the
aminoacyl-tRNA (A site) and the elongating peptide
chain (P site)
E) The 50S subunit is a part of the initiation complex
During RNA synthesis, the DNA template sequence TAGC
would be transcribed to produce which of the following
sequences?
A. ATCG
B. GCTA
C. CGTA
D. AUCG
E. GCUA
The answer is . RNAis antiparallel and complementary to the
template strand. Also remember that, by convention, all base
sequences are written in the 5' to 3' direction regardless of the
direction in which the sequence may actually be used in the
cell.
Elongation factors are targets of bacterial
toxins (eg, Diphtheria, Pseudomonas).
Which of the following is true regarding the
so-called caps of RNA molecules?
a. They allow correct translation of
prokaryotic mRNA
b. They are unique to eukaryotic mRNA
c. They allow tRNA to be processed
d. They occur at the 3′ end of tRNA
e. They are composed of poly A
