2. Structure of a typical
Structure of a typical
mRNA
mRNA
7MeG
3. The genetic code (U=RNA, T=DNA)
“wobble”
position
first base
second base
4. There are 64 codons for 20 amino acids
first base
“wobble”
position
5.
6.
7. Messenger RNA (mRNA) is transcribed
from DNA (pre-mRNA) and processed
(spliced) in the nucleus
The machinery for translating mRNA is
the ribosome, a large 2 subunit complex
of proteins and ribosomal RNA (rRNA)
8. transfer RNA: (>1
for each aa) the
transporter of
amino acids to the
ribosome
antiparallel
binding - like
DNA
9. Aminoacyl tRNA synthetases
one for each aa important role in fidelity of translation
(selectivity for correct aa; hydrolysis of incorrect aa-tRNA)
(Adenylated aa)
Mutated in cancers, neuropathies, autoimmunity, metabolic disease
11. is the process that takes the information passed from DNA as messenger
RNA and turns it into a series of amino acids bound together with peptide
bonds. It is essentially a translation from one code (nucleotide sequence) to
another code (amino acid sequence). The ribosome is the site of this action,
just as RNA polymerase was the site of mRNA synthesis. The ribosome
matches the base sequence on the mRNA in sets of three bases (called
codons) to tRNA molecules that have the three complementary bases in their
anticodon regions. Again, the base-pairing rule is important in this
recognition (A binds to U and C binds to G). The ribosome moves along the
mRNA, matching 3 base pairs at a time and adding the amino acids to the
polypeptide chain. When the ribosome reaches one of the "stop" codes, the
ribosome releases both the polypeptide and the mRNA. This polypeptide will
twist into its native conformation and begin to act as a protein in the cells
metabolism.
Translation
12. A protein is being translated from an mRNA with the sequence
5' augcaaucauaugcuucugcuauguuaagc.. 3’.
Starting with aug, the first five amino acids of the peptide are:
Met - Gln - Ser - Tyr - Ala
Figure 1. Summary of transcription and translation showing the relationship of DNA
and RNA nucleotides with amino acids
13. The Steps of Translation
A ribosome is an intercellular structure made of both RNA and protein, and
it is the site of protein synthesis in the cell. The ribosome reads the
messenger RNA (mRNA) sequence and translates that genetic code into a
specified string of amino acids, which grow into long chains that fold to
form proteins.
Figure 3. Summary of ribosome structure and positions of E, P, and A sites
14. Remembering what happens in the E, P, and A sites:
P = peptidyl because the P site holds the tRNA with the
growing peptide
A = aminoacyl because the A site holds the incoming
amino acid that will accept the transfer of the peptide
from the P site
E = exit site because the tRNA from the P site will exit the
ribosome through this site after peptide bond formation
and translocation
15. It is the first stage in protein synthesis, is the
process of assembly of large and small ribosomal
subunits to form an ribosome containing initiator
transfer RNA (tRNA) (Met-tRNAiMet) that is base
paired to the initiation codon of messenger RNA in
the ribosomal peptidyl (P) site.
Initiation
16. The small subunit binds to a site upstream (on the 5' side) of the
start of the mRNA. It proceeds to scan the mRNA in the 5'-to-3'
direction until it encounters the START codon (AUG). Nearby
sequences help position the small subunit. In prokaryotes, this
sequence, called the Shine-Dalgarno sequence, is upstream of the
AUG. In eukaryotes, a consensus sequence (Kozak sequence)
surrounds the AUG.
Figure 4. During Initiation
17. - is a key step of protein synthesis, during which the
nascent polypeptide chain extends by one amino acid
residue during one elongation cycle.
- also elongation requires specific aminoacyl-tRNAs
being escorted to the ribosome by GTP- coupled
elongation factor. It also requires movement along the
ribosome-coupled mRNA, three nucleotides at a time
to add aminoacids that have been bound to tRNAs.
Elongation
18. A tRNA bound to its amino acid (known as an aminoacyl-tRNA) that is able to base
pair with the next codon on the mRNA arrives at the A site. The ribosome catalyzes
the formation of a peptide bond between the preceding amino acid (Met at the start
of translation) in the P site and the amino acid held by the tRNA in the A site. The
initiator tRNA moves to the E site and the ribosome translocates (moves down) one
codon downstream. This shifts the more most recent tRNA from the A site to the P site,
opening up the A site for the arrival of a new aminoacyl-tRNA.
Figure 5. During elongation
19. - termination happens when a stop codon in the
mRNA (UAA, UAG, and UGA) enters the A site. Stop
codons are recognized by proteins called release
factors, which fit neatly into P site ( through they
aren’t tRNA).
Termination
20. There are no tRNA molecules with anticodons complementary to stop
codons, instead protein release factors (RF) recognize these codons when
they arrive at the A site. Binding of a release factor causes the polypeptide
(protein) to be released from the ribosome. The ribosome subunits
dissociate (split) from each other and can be reassembled later for another
round of protein synthesis.
Figure 6. During termination
21. Polyribosomes
Ribosomes do not work singly on a mRNA
but in multiple copies on the mRNA – a
polyribosome – like a string of beads
22. Translation speed
• Translation speed of each ribosome = 15 amino
acids/sec
• Multiple ribosomes processing simultaneously
a 300 a.a. long protein, i.e. one ribosome every
30a.a. of synthesized protein - the number of
protein molecules produced in 1 min is ~4000
29. Post-translational modification
Only 20 amino acids – cell uses post-
translational modifications (over 200) to
increase diversity, including:
Disulphide bond formation (e.g. insulin)
Proteolytic cleavage (e.g. insulin -> A and B
chains)
Addition of carbohydrate (Glycosylation)
Addition of phosphate (Phosphorylation)
Addition of lipid groups (Prenylation, Acylation)
Hydroxylation (e.g. Collagen; Leitinger lecture)
30. Insulin biosynthesis
in pancreatic β cells
Insulin undergoes
extensive post-
translational modification
along the production
pathway, including
disulphide bond formation
in the ER and proteolytic
cleavage in the secretory
vesicle to produce active
insulin
31. Glycosylation in the RER and Golgi complex
Pre-assembled carbohydrate
chains N-linked to Asn of
(AsnXSer/Thr) “sequon”
Carbohydrate chains processed
by trimming followed by
extension
