RNA TRANSLATION

Protein Synthesis
 Protein synthesis-Translation
• Overview
• Genetic information in chromosomes is
transmitted to daughter cells (replication)
→ mRNA → translations → protein
• the above is called the “central dogma”
• exceptions are certain RNA viruses
• translation requires a genetic code
• alteration in nucleic acid sequence →
mutation → wrong amino acid in position in
protein → disease or death
 The Genetic Code

• genetic code is a dictionary: triplet of nucleotides

→ amino acid
• words are called codons
• A. Codons
• codons presented in mRNA bases of A, G. C
and U
• sequence always written from 5’→3’ end
• 4 bases used to produce triplet codons
• triplet combinations of 4 bases → 64
combinations
The Genetic code
 How to translate a codon
– The genetic code table can be used to translate any codon
sequence
– E.g. CAU codes for histidine
– AUG → methionine
– 61 of 64 codons code for the 20 common amino acids
– Termination (“stop” or “nonsense”) codons:
– 3 codons: UAG, UGA and UAA function as termination
codons
– do not code for any amino acid
– signal termination of translation
– Consequences of altering the nucleotide sequence:
– Changing a single base in mRNA sequence = point
mutation → 3 possible results
 • Silent mutation:
» Changed codon may code for same amino
acid
» UCA → UCU → same amino acid, Serine
» Called “silent” mutation
• Missense mutation:
» Changed codon may code for different
amino acid
» UCA → CCA = Ser → Pro
• Substitution of different amino acid → missense
mutation
• Nonsense mutation:
» UCA → UAA = Ser → termination codon
» Causes premature termination of sequence
= nonsense mutation
 Characteristics of the genetic
code
• Genetic code quite constant throughout all
organisms
• Assumed that one standard code evolved in
primitive organisms
• Adoption of the genetic code = “accident frozen in
time”
• Characteristics of code:
• Specificity:
– Specific – a specific codon always codes for same
amino acid
• Universality:
– Code is universal i.e., with few exceptions, it has
been conserved across different organisms
• Redundancy:
– Code is degenerate
– Each codon → single amino acid but
– One amino acid may have more than 1 codon coding
for it
– E.g. Arg coded for by 6 different codons

• Nonoverlapping and commaless:

– Code is read from a fixed starting point as continuous
sequence of bases, 3 at a time
– If one or more are deleted, → frame-shift mutation
and reading frame is altered → very different amino
acids being coded for from that point on
– If 3 or multiples of 3 bases are added/deleted, there is
no frame shift, just the addition/deletion of the
specified a.a.
 Components Required for
Translation
• Large number of components are required for
synthesis of polypeptide chains
• All the amino acids, the mRNA, the tRNAs,
functional ribosomes, energy sources, enzymes
and protein factors needed for initiation,
elongation and termination
• These all gather in the cytosol prior to synthesis
Amino acids
• All amino acids to be incorporated into the
protein must be present in the cytosol
• If one is missing due to insufficiency in diet or
some synthesis disorder, translation of the
mRNA stops at that codon
• Transfer RNA (tRNA)

• At least one type of tRNA is required per amino acid

• There are at least 50 species of tRNA in humans while
bacteria have 30 – 40
• Since only 20 different amino acids, some amino acids have
more than one tRNA
• Especially those coded for by more than 1 codon

• Amino acid attachment site:

– Each tRNA has an attachment site for a specific amino

acid at its 3’-end
– When a tRNA has a covalently attached amino acid, it is
charged
– Amino acid attached is said to be activated
• Anticodon:
– Each tRNA also has a 3-base sequence –
anticodon to recognize a specific codon
– This codon specifies the need for that amino
acid in the growing peptide
– tRNA are known as adaptor molecules
because they bind to the mRNA at their
anticodon end and bring the specified amino
acid at their 3’-end
• Messenger RNA (mRNA)
• the specified mRNA for the polypeptide to be
synthesized must be present as a template
• Aminoacyl-tRNA synthetases
• this is a family of enzymes required for
attachment of amino acids to their tRNAs
• each member of the family recognizes a specific
amino acid and tRNA
• each aminoacyl-tRNA synthetase catalyzes a 2-
step reaction → covalent attachment of amino
acid to 3’ end of tRNA
• reaction requires ATP → AMP + PPi
• the aminoacyl-tRNA synthetase is highly specific
and responsible for fidelity of translation
• Functionally competent ribosomes
• ribosomes are large complexes of protein
and rRNA
• made up of 2 subunits – large and small
• sizes are stated in Svedberg values (S)
• S units are determined by both shape and
molecular mass and are not additive
• Prokaryotic 70S ribosome is made up of
large 50S and small 30S unit
• Both prokaryotic and eukaryotic ribosomes
are similar in structure and serve as protein
manufacturing factories
• Ribosomal RNA (rRNA):
– Prokaryotic ribosomes contain 3 molecules of
rRNA while eukaryotic ribosomes have 4 rRNAs
– have extensive secondary structure
comparable to that found in tRNA

• Ribosomal proteins:
– Are present in greater numbers in eukaryotic
ribosomes than in prokaryotic
– Play number of roles in structure and function of
ribosomes
• A and P sites on the ribosome:
– Ribosome has 3 binding sites for tRNA molecules
– The A ,P and E sites extend over both subunits
– During translation, the A site binds the incoming
aminoacyl-tRNA according to the codon at that site
– At the same time, the P site carries the peptidyl-
tRNA chain already synthesized
– The E site holds the existing (uncharged) t-RNA
• Cellular location of ribosome:
– In eukaryotic cells, ribosomes are found either free
in the cytosol or
– On the surface of ER – called rER
– Those on ER synthesize proteins destined either for
export outside the cell or to be incorporated into
cell membranes of different organelles
• Mitochondria have their own ribosomes
• Protein factors
• Factors needed for protein synthesis include
those for
• Initiation
• Elongation
• Termination
• Some have a catalytic function, others
stabilize the synthetic machinery
• ATP and GTP are required as sources of
energy
• To add 1 amino acid to a peptide chain, 4
high-energy bonds must be cleaved:
• 2 from ATP in aminoacyl-tRNA synthetase
reaction (1 in removing PPi, the other in
converting PPi → 2Pi by pyrophosphatase)
• 2 from GTPs (one for binding aminoacyl-tRNA
to the A site, and one for translocation
• Additional ATP and GTP are required for
initiation and termination
Codon Recognition by tRNA
• Recognition of a particular codon in mRNA is accomplished
by the anticodon sequence
• Some tRNAs recognize more than one codon

Antiparallel binding between codon and anticodon

• Binding of tRNA to mRNA follows the rule of complementary
binding:
• mRNA read 5’→3’ direction

“Wobble” hypothesis
• “wobble” means that tRNAs can recognize more than one
codon
• the hypothesis states that the base at the 5’ end of the
anticodon (3’-end of codon) is not as spatially defined as
other two bases
• movement of that first base allows nontraditional binding n
• therefore need not be 61 tRNA species corresponding to the
61 codons
 Wobble
hypothesis
 Steps in Protein Synthesis
• protein synthesis pathway is called “translation” because
mRNA is translated into polypeptide sequence
• mRNA from 5→3’ end is translated to protein from N- to
C- end

• prokaryotic mRNA often have several coding regions –

they are polycistronic
• each coding region has its own initiation codon and
produces a separate polypeptide

• eukaryotic mRNA is monocistronic

• translation is divided into initiation, elongation and
termination
• polypeptides may be subjected to post-translational
modification
• eukaryotic and prokaryotic protein synthesis are similar in
most details
• steps of protein synthesis in E. coli are shown
 Initiation
• involves assembly of components before
peptide bond formation occurs
• components are:
• 2 ribosomal subunits
• mRNA
• aminoacyl-tRNA specified by first codon
• GTP – provides energy
• Initiation factors that facilitate assembly of
the whole initiation complex
• In prokaryotes, 3 initiation cofactors
known: IF-1, IF-2 and IF-3
• In eukaryotes, 10 known – designated eIF
• 2 mechanisms by which ribosomes recognize
nucleotide sequence
• Shine-Dalgarno sequence:
– In E. coli, the Shine-Dalgarno sequence is 5’-
UAAGGAGG-3’
– Located 6 – 10 bases upstream of AUG codon
– 16S rRNA component of 30S subunit has a sequence
near its 3’ end complementary to nearly all of the
Shine-Dalgarno sequence
– mRNA 5’-end and 3’-end of 16S rRNA can form
complementary base pairs → binding and positioning
of mRNA on 30S ribosomal subunit
 Initiation codon

– AUG is recognized by a special initiator rRNA

– Recognition is by IF-2 in E. coli and eIF-2 in humans
– In bacteria and mitochondria, initiator tRNA carries N-
formyl methionine
– Formyl group added to methionine after the amino acid
is attached to initiator tRNA by transformylase – uses
N10-formyl tetrahydrofolate as donor
– In eukaryotes, initiator tRNA carries methionine – not
formylated
– In both prokaryotes and eukaryotes, the Met is
removed before the protein is completed
 Elongation
• Involves adding amino acids to carboxyl end of growing
polypeptide chain

• During elongation, ribosome moves from 5’-end to 3’-end of

mRNA being translated

• Delivery of the aminoacyl-tRNA is facilitated in E. coli by

elongation factors EF-Tu and EF-Ts and requires GTP
• In eukaryotes, comparable elongation factors are designated
eEF’s.

• Formation of peptide bonds catalyzed by peptidyltransferase –

activity intrinsic to 23S rRNA in 50S ribosomal subunit)

• Because rRNA can act as an enzyme, it is called a ribozyme

 Translocation
• After peptide bond formation, ribosome
advances 3 positions toward 3’-end of
mRNA: translocation

• This requires elongation factor EF-G and

GTP in E. coli and is similar in eukaryotes
• Causes release of uncharged tRNA and
movement of the peptidyl-tRNA into the P
site.
 Termination
• Involves 1 of 3 termination codons moving into the A site
• Codons are recognized in E. coli by release factors
• RF-1 recognizes UAA and UAG
• RF-2 → UGA and UAA
• RF-3 binds GTP and stimulates activity of RF-1 and
RF-2
• The factors → newly synthesized protein to be released
from ribosomal complex + dissociation of ribosome from
mRNA
• eukaryotes have a single release factor eRF that also
binds GTP
• newly synthesized protein may undergo modification
• mRNA, tRNA, protein factors and
ribosomal subunits are recycled to
synthesize another protein
• some inhibitors of protein synthesis are
shown:
• Antibiotics
• Streptomycin
• Tetracyclines
• Puromycin
• Chloramphenicol
• Clindamycin & Erythromycin
Polysomes

• more than one ribosome at a time can be

involved in translating the mRNA into protein.
• The complex of one mRNA and several
ribosomes → polysome or polyribosome
 Post Translational
Modification of Polypeptides
 Post-Translational Modification of
Polypeptide Chains

• many polypeptides are covalently modified

while still attached to ribosome
• called post-translational modifications
because it occurs after protein synthesis is
initiated
• may involve removal or covalent addition of
chemical groups
Trimming
• many proteins for secretion originally synthesized as large
precursor molecules – not functionally active

• portions of the protein must be cleaved by specialized

endoproteases → release of active molecule

• various cellular sites of cleavage of newly formed proteins

e.g. some precursor proteins cleaved in ER or Golgi others
cleaved in developing secretory vesicles (e.g. proinsulin to
insulin).
• Collagen cleaved after secretion

Zymogens = inactive precursors of secreted enzymes

(including digestive proteases)

• become activated through cleavage once in their proper site

of action e.g. pancreatic zymogen, trypsinogen → trypsin in
small intestine
Covalent modification
both enzymatic and structural proteins → activated or inactivated by covalent
attachment of chemical groups:

Phosphorylation:
– Occurs on –OH group of Ser, Thr or less frequently
Tyr
– Catalyzed by one of a family of protein kinases
– Reversed by action of cellular protein
phosphatases
– Phosphorylation → ’se or ’se in activity
Glycosylation:

– Proteins destined → plasma membrane,

lysosomes or to be secreted have
carbohydrate chains attached to Ser or Thr –
OH groups (O-linked) or asparagines (N-
linked)
– Stepwise addition of sugars occurs in ER and
Golgi
• Hydroxylation:
– Pro and Lys residues on -chains of collagen →
extensively hydroxylated in ER

– Other covalent modifications:

– E.g. vitamin biotin may be covalently bound to
protein component of carboxylase enzyme to be
catalytically active.
– Acylation- membrane anchoring
– Caboxylation of glutamate residues on clotting
factors- vitamin K -dependent
– Farnesylation- membrane insertion of proteins-
oncogenes