Cell and Molecular Biology Module

1. Dr Zahid Azeem
Assistant Professor
Biochemistry
AJK Medical College, Muzaffarabad
MBBS-2019--- CMB Module

2. RNA for translation
Exists in 3 forms:
mRNA (messenger) made by eukaryotic RNAP II
tRNA (transfer) made by eukaryotic RNAP III
rRNA (ribosomal) made by eukaryotic RNAP I
made (transcribed) in the nucleus, used in the
cytoplasm
Single stranded

4. Genetic Codes
Specificity
Universality
Degeneracy
Nonverlapping and Commaless

5. mRNA
RNA nucleotides transcribed from DNA
Codons: mRNA nucleotide triplets that code for
amino acids

6. tRNA
tRNA carries the amino
acid to make the
polypeptide chain
Anticodon:
complementary sequence
on tRNA
Fig. 17.12
Codon

7. tRNA Structure
Hydrogen bond base pairing
between nucleotides of the
same single strand of RNA (80
nucleotides)
Fig. 17.13
Cloverleaf
L-shape
Anticodon loop:
H-bonds with
mRNA codon
3’ end
3’ end

8. tRNA Activation
Enzyme aminoacyl-tRNA
synthetase
 attach appropriate amino acid to
tRNA
 catalyze covalent joining of
amino acid to tRNA
 tRNA + amino acid = aminoacyl-
tRNA (aatRNA)
20 different aatRNA synthetases
for each of the 20 different
amino acids
tRNA molecule is reactivated
many times (recycled)
Fig. 17.14
http://www.bio.davidson.edu/courses/genomics/2005/drysdale/molecular%20function.jpg

9. Ribosome Structure
2 subunits:
large (60S) and small (40S)
Final size is 80S
S (Svedberg): a unit of
measure of size based on
how quickly an object
sediments in a centrifuge

10. Ribosome Binding Sites
Ribosome binds mRNA
binding site
3 tRNA binding sites
E, P, A

11. Ribosome tRNA binding sites
A site:
aminoacyl-tRNA site
holds the aatRNA carrying the next
amino acid to be added
P site:
peptidyl-tRNA site
holds the tRNA molecule carrying
the growing polypeptide chain
E site:
Exit site
where tRNA molecules leave the
ribosome

12. Translation Mechanism
Initiation
Elongation
Termination
Post-translational modifications

13. Initiation step 1: Ribosome finds and
binds to the mRNA strand
Prokaryotes:
mRNA transcript has a Shine-Dalgarno sequence
upstream of initiation AUG codon
16S rRNA on ribosome small 30s subunit has a
complementary sequence: anti Shine-Dalgarno sequence
Eukaryotes
Ribosome small subunit recognizes and bind to mRNA at 5’
cap

14. Initiation step 2: Ribosome locates
translation start site
Ribosome small subunit moves along 5’ leader of
mRNA until reach translation start site (start codon
AUG)
Factors that help ribosome small subunit find start
codon:
Prokaryotes: Initiation factors
Eukaryotes: Kozak sequence on the mRNA
Fig. 17.8

15. Initiation step 3: Initator tRNA binds
Once ribosome
small subunit reach
AUG, initiator
tRNA attaches
AUG
H bonds forms
between the
mRNA codon and
tRNA anticodon

16. Initiation step 3: Initator tRNA binds
Starting amino acid:
Prokaryotes: formyl-methionine (fmet)
Eukaryotes: regular methionine (met)

17. Initiation step 4: Ribosome large
subunit binds
= complete ribosome
+ initiator tRNA
+ mRNA at AUG
Forming
complex
requires
energy
Final position of
initiator tRNA is
in P site

18. Elongation Step 1:
Codon recognition
Incoming aa-tRNA to A
site
H bonds form between
the mRNA codon and
tRNA anticodon
Energy is required

19. Elongation Step 2:
Peptide bond formation
Ribosome catalyzes the formation of
a peptide bond
between the amino acid in the P-site to
the amino acid in the A-site
involves the carboxyl end of the
polypeptide chain
Result:
polypeptide chain is longer by one
amino acid
polypeptide chain is transferred to
tRNA at the A site

20. Elongation Step 3: Translocation
Translocation requires
energy
Ribosome moves:
tRNA from P site to E site:
leaves ribosome
tRNA from A site to P site:
polypeptide returns to P site,
ready for next polymerization
A site is now empty
next aatRNA can bind

22. Unidirectional Translocation
mRNA bound to tRNA
Translocates by 1 codon (3
nucleotides)
Ribosome reads mRNA 5’  3’
mRNA moved through ribosome
unidirectionally

24. Termination
At stop codon a protein called release factor binds to A site
(no tRNA for stop codon, thus no aatRNA)
Release factor:
adds water molecule instead of amino acid to polypeptide
polypeptide hydrolyzed from tRNA in P site and released
Translation complex disassembles
Fig. 17.19

26. Wobble Mechanism
tRNA can recognize more than one codon for
particular amino acid- Wobble hypothesis
First base of tRNA pairs non-stringently
Hence, no need of 61 tRNA for 61 codons

27. Wobble standards for Non-
standard pairing

28. Polyribosomes
A single strand of mRNA can be used to make
many copies of a polypeptide simultaneously
Polyribosomes: when 1 molecule of mRNA has
multiple ribosomes simultaneously translating the
mRNA
Fig. 17.20

29. Protein Synthesis in Prokaryotes
Prokaryotes don’t
have a nucleus
How does that
change protein
synthesis?
Fig. 17.22

30. Post-Translational Modifications
Addition: of sugars, lipids, or phosphate groups
Removal (Cleavage): of some amino acids (such
as the methionine) or whole polypeptide chains.
Polymerization: Two or more polypeptides may
join to form a protein. Example: hemoglobin
Folding

31. 1- Triming

32. Example: Insulin

33. Covalent Modification
Phosphorylation
Glycosylation
Hydroxylation
Other covalent modification; that include
biotinylated enzymes and carboxylation

34. Protein folding
Molecular chaperones
Gro ES and Gro EL
Maintaining native confirmation
Protein degration
Ubiquitinylation
Proteosome
E1, E2, E3

35. THE END