Basic Molecular Biology: The Genetic

Process

Translation

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Concepts to remember in translation

Degeneracy of the triplet code

Adaptor hypothesis
tRNA structure and ribosome assembly
Overview of polypeptide synthesis
Post-translational modifications

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 A processed mRNA ready for translation

5’ untranslated 3’ untranslated
region region

Protects from degradation/ Protects from degradation/

recognition for ribosome transport to cytoplasm
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What is the pathway from DNA to Protein

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How is RNA used to produce a protein?-- translation
mRNA directed synthesis of polypeptides
Translates DNA sequence information into proteins
Genetic code dictates translation of specific RNA triplet codons to amino acids
Occurs in the cytosol

A polysome

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 Key concepts in translation
Genetic information transcribed from DNA to mRNA as a
nonoverlapping, degenerate triplet code
1 codon = 1 amino acid but 1 amino acid > 1 codon
 Triplet code AUGAGGCAAGCAUGCACGCAAGAAGGGCAGCAAAA
 Degenerate –
more than 1
triplet may
encode same
amino acid
 Non overlapping
E.g AUGCGTACT

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 Adaptor hypothesis
tRNA acts as a ‘shuttle’ linking amino acid to
nucleic aid
Aligns correct amino acids to form a polypeptide
One tRNA per amino acid

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 What is the structure of tRNAs?
class of small RNAs which form covalent bonds to amino acids
allows correct insertion of amino acids into the elongating
polypeptide chain.

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How many tRNAs are needed to translate all
the codons?

How is the correct reading

frame identified? 3 base anticodon in tRNA
allows base-pairing with corresponding
sequence in mRNA

AGGCAAGCAUGCACGC 9
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 Translation has 2 important recognition steps
1 Correct aminoacylation (‘charging’): Covalently attach the correct
amino acid to tRNA (specified by anticodon)
2 Select the correct charged tRNA as specified by mRNA

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2 Select the correct charged tRNA as specified by mRNA
• Ribosomes select aa-tRNA
based only on their codon –
anticodon interactions
• This pairing is antiparallel and
the base in the third position
forms non standard base
pairing (Wobble hypothesis)

tRNA anticodon 3’-A A G-5’ or 3’-A A G-5’

mRNA codon 5’-U U C-3’ 5’-U U U-3’

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Translation

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 Key concepts in translation
Genetic information transcribed from DNA to mRNA as a
nonoverlapping, degenerate triplet code
1 codon = 1 amino acid but 1 amino acid > 1 codon
2 key molecules responsible for decoding nucleotide
sequence into amino acid sequence are tRNAs and
aminoacyl-tRNA synthetases
3 base anticodon in tRNA allows base-pairing with
corresponding sequence in mRNA
20 specific aminoacyl-tRNA synthetases present
Both pro and eukaryotic ribosomes have a large and small
subunit
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 Translation requires…..
1) mRNA
2) Aminoacyl- transfer RNA (aatRNA)
3) Ribosomes

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Translation requires…..

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Ribosomes

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 Ribosomes
Made of
rRNA &
ribosomal proteins
2 subunits
– large
- small

Subunits are self assembling

combine only in the presence of mRNA and a charged
(aminoacylated) tRNA
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 Polypeptide synthesis (overview)

3 distinct steps

1. Chain initiation
2. Chain elongation
3. Chain termination

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Polypeptide synthesis (overview)

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 Initiation
in
eukaryotes

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 Initiation
in
Prokaryotes

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 Chain elongation
4 stage reaction cycle
1) aatRNA binding
aatRNA binds to A site on ribosome by base pairing with codon
2) Conformation change in ribosome: induced by GTP hydrolysis
of EF1
3) Transpeptidation
C terminal of polypeptide uncoupled from P site tRNA and
peptide bond transferred to amino acid on A site tRNA
catalysed by peptidyltransferase
4) Translocation
GTP hydrolysis of EF2 causes 2nd conformational change P site
tRNA is transferred to E site
Simultaneous transfer of A site tRNA moved to P site
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How does elongation proceed?

Which end of the protein is produced first?

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 Chain
elongation

4 Steps

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Chain elongation

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 termination
Release factors (eRFs) recognise
and bind to stop codons

This induces peptidyl transferase

to transfer peptidyl group to
water instead of aatRNA
Uncharged tRNA released from
ribosome
Inactive ribosome then release
mRNA

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termination

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 Release Factors
• Prokaryotic translation termination is mediated by 3
factors:
– RF1 recognizes UAA and UAG
– RF2 recognizes UAA and UGA
– RF3 is a GTP-binding protein facilitating binding of RF1
and RF2 to the ribosome
• Eukaryotes has 2 release factors:
– eRF1 recognizes all 3 termination codons
– eRF3 is a ribosome-dependent GTPase helping eRF1
release the finished polypeptide

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Dealing with Aberrant Termination
• Two kinds of aberrant mRNAs can lead to aberrant
termination
– Nonsense mutations can occur that cause premature
termination
– Some mRNAs (non-stop mRNAs) lack termination codons
• Synthesis of mRNA was aborted upstream of termination codon
• Ribosomes translate through non-stop mRNAs and then stall
• Both events cause problems in the cell yielding
incomplete proteins with adverse effects on the cell
– Stalled ribosomes out of action
– Unable to participate in further protein synthesis

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 Non-Stop mRNAs
• Prokaryotes deal with non-stop mRNAs by transfer-
messenger RNAs (tmRNA)-mediated ribosome rescue
– Alanyl-tmRNA resembles alanyl-tRNA
– Binds to vacant A site of a ribosome stalled on a non-stop
mRNA
– Donates its alanine to the stalled polypeptide
• Ribosome shifts to translating an ORF on the tmRNA
(transfer-messenger RNA)
– Adds another 9 amino acids to the polypeptide before
terminating
– Extra amino acids target the polypeptide for destruction
– Nuclease destroys non-stop mRNA
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 Non-Stop mRNAs
• Prokaryotes deal with
non-stop mRNAs by
tmRNA-mediated
ribosome rescue
– tmRNA are about 300
nt long
– 5’- and 3’-ends come
together to form a
tRNA-like domain (TLD)
resembling a tRNA
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Figure 18.31 Mechanism of tmRNA-mediated release of non-stop mRNA and
polypeptide. (a) EF-Tu, alanyl-tmRNA and SmpB (turquoise) bind to the A site of the
ribosome stalled on a non-stop mRNA (brown). SmpB helps the tRNA-like domain of the
tmRNA bind to the ribosome. (b) The ribosome’s peptidyl transferase transfers the
alanine (yellow) from the tmRNA to the stalled polypeptide (green). (c) The ribosome
shifts to reading the ORF (purple) of the tmRNA. (d) The ribosome completes
translating the ORF of the tmRNA, adding nine more amino acids (red) to the end of
the stalled polypeptide and releasing it. (e) Together, these extra amino acids target
the whole polypeptide for destruction. At the same time, the non-stop mRNA is
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destroyed, perhaps by RNase R, which associates with tmRNA.
 Eukaryotic Aberrant Termination
• Eukaryotes do not have tmRNA
• Eukaryotic ribosomes stalled at the end of the
poly(A) tail contain 0 – 3 nt of poly(A) tail
– This stalled ribosome state is recognized by carboxyl-
terminal domain of a protein called Ski7p
– Ski7p also associates tightly with cytoplasmic exosome,
cousin of nuclear exosome
– Non-stop mRNA recruit Ski7p-exosome complex to the
vacant A site
– Ski complex is recruited to the A site
• Exosome, positioned just at the end of non-stop
mRNA, degrades that RNA
• Aberrant polypeptide is presumably destroyed
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 Exosome-Mediated Degradation

Figure 18.32 Model for exosome-mediated degradation of eukaryotic non-

stop mRNA. (a) The A site of a ribosome stalled at the end of a non-stop
mRNA (brown) contains zero to three nucleotides of the mRNA’s poly(A) tail.
Here, no A’s are in the A site. This state of the ribosome is attractive to the
Ski7p–exosome complex (yellow and red), which binds to the vacant A site. (b)
Next, the Ski complex (purple) binds to the A site, and (c) this triggers
degradation of the non-stop mRNA and release of the ribosomal subunits.

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 Premature Termination
NAS (nonsense-associated
altered splicing)

NMD (nonsense-
mediated mRNA
decay)

Figure 18.33 Models for NAS and NMD. (a) NAS. Upf1, perhaps in conjunction with other
proteins, senses a premature stop codon in the reading frame of the future mRNA and induces an
alternative splicing pattern (purple) to produce the mature mRNA at top, which lacks the premature
stop codon. (b) Standard splicing (orange) produces a mature mRNA with a premature stop
codon, and Upf1 and Upf2 bound at the exon/exon boundaries. (c) NMD. Upf1 and Upf2 (brown
and gray), perhaps in conjunction with other proteins, sense the in-frame premature stop codon
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too
close to the second exon/exon boundary and induce destruction of the mRNA.
 Premature Termination
• Eukaryotes deal with premature termination
codons by 2 mechanisms:
– NMD (nonsense-mediated mRNA decay)
• Mammalian cells use a downstream destabilizing
element
• Yeast cells appear to recognize a premature stop codon
– NAS (nonsense-associated altered splicing)
• Senses a stop codon in the middle of a reading frame
• Changes the splicing pattern so premature stop codon
is spliced out of mature mRNA
– Both mechanisms require Upf1

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 Mammalian NMD
NMD in mammalian cells involves a downstream
destabilizing element
• Upf1
• Upf2
– Bind to mRNA at exon-exon junction that measures
distance to a stop codon
– Codon far enough upstream
• Looks like a stop codon
• Activates downstream destabilizing element to degrade
mRNA
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NAS and NMD Models

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 Posttranslation
• Translation events do not end with
termination
– Proteins must fold properly
– Ribosomes need to be released from mRNA and
engage in further translation rounds
• Folding is actually a cotranslational event
occurring as nascent polypeptide is being
made

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 Folding Nascent Proteins
• Most newly-made polypeptides do not fold
properly alone
– Polypeptides require folding help from molecular
chaperones
– E. coli cells use a trigger factor
• Associates with the large ribosomal subunit
• Catches the nascent polypeptide emerging from ribosomal
exit tunnel in a hydrophobic basket to protect from water
– Archaea and eukaryotes lack trigger factor, use
freestanding chaperones

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Release of Ribosomes from mRNA
• Ribosomes do not release from mRNA
spontaneously after termination
• Help is required from ribosome recycling
factor (RRF) and EF-G
– RRF resembles a tRNA
• Binds to ribosome A site
• Uses a position not normally taken by a tRNA
– Collaborates with EF-G in releasing either 50S
ribosome subunit or whole ribosome
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Post translational modifications
Protein folding
• Nascent protein is folded and/or modified into functional forms
• Amino acid sequence determines its folding into specific 3-D conformation
• This folding is mediated by molecular chaperones (e.g. Hsp70) or chaperonins
(Hsp60 complexes)

Covalent modification
• Various chemical groups (e.g acetyl, phosphoryl, hydroxyl, glycosyl etc) are
added to the NH2 or COOH terminal or internal residues of the polypeptide
• These modifications are essential and dictate the activity, life span or the
cellular location of proteins.

Proteolytic cleavage
Activates some inactive precursors
E.g. caspases, zymogens etc

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 Death of proteins

Proteins that are misfolded, denatured, in excess or extracellular in

origin are targeted for degradation within lysosomes

Another pathway is by the addition of ubiquitin to lysine residues,

which targets it for destruction by the proteosome complex.

Degradation of proteins can be a part of normal cell processes (cell

cycle) or may be implicated in disease, especially
neurodegenerative diseases (Parkinsons, Alzheimers)

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Death of proteins

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Lysosomes Degredation

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Proteosomal Degredation

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Some proteins function in the cytoplasm; others need to be
transported to various organelles.

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How can proteins be delivered to
their appropriate destinations?

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Proteins are directed
to their destinations
via signals in the
amino acid sequence

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