post translational Modification

2. Posttranslational modification (PTM) is a step in protein
biosynthesis.
Proteins are created by ribosomes translating mRNA into
polypeptide chains.
polypeptide chains undergo PTM (such as folding, cutting and
other processes) before becoming the mature protein product.
modification of amino acids extends the range of functions of the
protein by attaching it to other biochemical functional
groups (such
as acetate, phosphate,various lipids and carbohydrates), changing
the chemical nature of an amino acid , or making structural
changes (e.g. formation of disulfide bridges).

3. Processing and
Folding
Post-
translational
modification

4. Post-
translational
modification
Proteolytic cleavage
Modification of aminoacids
Subunit aggregation
Protein folding and chaperones

5. Modification Location
Proteolysis
Signal peptide cleavage
Mitocondria
Chloroplasts
Endoplasmic
Recticulum
Proteolysis
Processing
Cytoplasm
Secretory Vesicles
Acylyation Cytoplasm
Myristoylization Cytoplasm
O Glycosolation Cytoplasm
Golgi Apparatus
Palmitoyl Addition Cytoplasm
What Happens Where

6. Palmitoyl Addition Cytoplasm
Virus Processing Cytoplasm
Glycosolation of Asn Endoplasmic
Recticulum
Palmitoyl and Glycosyl-
posphatidylinositolization
Endoplasmic
Recticulum
Post Carboxylation and
Hydroxylation
Endoplasmic
Recticulum
Disulfide bond Formation Endoplasmic
Recticulum
Modification of N-Glycosyl
groups
Golgi Apparatus
O-Glycosylation Golgi Apparatus
Sulfation of Tyr Golgi Apparatus
What Happens Where

7. Proteolytic cleavage

8. Proteolytic
cleavage

9. 4. Post-translational Quality Control: Selective proteolysis.
B. Ubiquitination requires 3 enzymes:
E1 (ubiquitin-activating enzyme) activates ubiquitin (U)
E2 (ubiquitin-conjugating enzyme) acquires U via high-energy thioester
E3 (ubiquitin ligase) transfers U to target proteins
Hierarchical organization: one or few E1s exist, more E2s, many E3s.
Other functions for ubiquitination (to be discussed in plasma membrane lecture).

10. 4. Post-translational Quality Control: Selective proteolysis
B. The Proteasome - high molecular weight (28S) protease complex that degrades
ubiquitinated proteins in the cytoplasm
Present in cytoplasm and nucleus, not ER
Uses ATP
Contains a 700 kD protease core and two 900 kD regulatory domains.
Highly conserved and similar to proteases found in bacteria.
Shaped like a cylinder.
Proteins enter the cavity, and are cleaved into small peptides.
Most but not all proteasome substrates are ubiqutinated.

11. Proteolytic cleavage

12. Modification of
amino acids

13. Modification of amino acids

14. Modification of aminoacids

16. Modification of amino acids

18. SUBUNIT AGGREGATION

20. PROTEIN
FOLDING AND
CHAPERONES

23. Transport from the ER to Golgi
 Appropriately modified proteins leave the ER and travel to
the Golgi Apparatus.
 They travel in membrane vesicles that arise from special
regions of membranes that are coated by proteins.
 There are of three types of coated vesicles that are well
characterized, clathrin-coated, COPI-coated and COPII-
coated vesicles.
 COPI and COPII act mainly in ER or Golgi cisternae.
 Clathrin acts in Golgi or plasma membranes.

24. The Golgi Apparatus has two major functions:
1. Modifies the N-linked oligosaccharides and adds O-linked
oligosaccharides.
2. Sorts proteins so that when they exit the trans Golgi network,
they are delivered to the correct destination.

26. Necessity of Gene Regulation
 Unicellular organisms
o Depending on the needs of the body some genes have to
be transcribed whereas the rest have to be switched off
o Helps to adapt to the changes in environment
 Multicellular organisms
 Helps in differentiation of cells
 Performance of various functions in the body

27. ►Gene Expression is Controlled by
Regulation Proteins: Activators and
Repressors
1.Activators, or Positive regulators, increase transcription of the regulated gene;
Repressors, or negative regulators, decrease or eliminate that transcription.
2. Many Promoters Are Regulated by Activation that Help RNA Polymerase Bind
DNA and by Repressors that Block that Binding.

28. Gene Regulation Mechanisms in Prokaryotes
and Eukaryotes
 In prokaryotes the primary control point is the process
of transcription initiation
 In eukaryotes expression of gene into proteins can be
controlled at various locations.

29. Gene Regulation in Prokaryotes
 Operon hypothesis- Proposed by two French
microbiologists, Francois Jacob and Jacques Monod in
1961 for which they won the nobel prize in 1965.
 Operon -In prokaryotes the linked genes are clustered
into units known as operons. The genes in a single
operon affect the same biochemical pathway that is
either they are expressed or repressed under similar
condition.
 Polycistronic RNA – In prokaryotes a single operon
gets transcribed into polycistronic mRNA which can
be translated into multiple proteins.

30. Control of Transcriptional Initiation
 Promoter sequences –Help the enzyme RNA polymerase
to recognize the transcriptional initiation sites
o -35 position is TTGACA
o -10 position is TATAAT
 Accessory or regulatory proteins –Control the ability to
recognize the transcriptional initiation sites
o Activators
o Repressors
 Operators - The interaction of the regulatory proteins
with the operators modulates the accessibility of promoter
regions of prokaryotic DNA.

31. Transcriptional Regulation in E.coli
 In E.coli the following two kinds of operons exist and in
both the cases the gene regulation is carried out by
repressor proteins.
o Catabolite-regulated operons -They are the operons which
produce gene products necessary for the utilization of energy.
Example lac operon.
o Attenuated operons – These operons produce gene
products necessary for the synthesis of small biomolecules
such as amino acids. These operons are typically attenuated
by the sequences within the transcribed RNA. Example trp
operon

32. The lac operon
 Components of lac operon
 Regulatory gene
o The regulatory gene is the i gene that codes for the repressor
protein of the lac operon.
o This i gene is expressed all the time hence it is also known
as a constitutive gene.
o The lac repressor protein has two functional domains or
regions one that binds the operator sequence and the other
that binds the lactose sugar.

33. Components of lac Operon Structural genes
o lac Z codes for β-galactosidase (β-gal), which is primarily
responsible for the hydrolysis of the disaccharide, lactose
into its monomeric units, galactose and glucose.
o Lac Y codes for permease, which transports lactose into
the cell.
o Lac A codes for transacetylase whose function is not
considered here
 Operator
 Promoter

34. Lactose operon: a regulatory gene and 3
stuctural genes, and 2 control elements
lacI
Regulatory gene
lacZ lacY lacA DNA
m-RNA
β -Galactosidase
Permease
Transacetylase
Protein
Structural Genes
Cis-acting elements
PlacI Plac
Olac
The LAC operon

35. lacY encodes a cell membrane
protein called lactose
permease to transport
Lactose across the cell wall
lacZ codes for β-galactosidase for
lactose hydrolysis
lacA encodes a thiogalactoside
transacetylase to get rid of
the toxic thiogalacosides
The LAC operon

36. i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
i p o z y a
b-Galactosidase
Permease
Transacetylase
Presence of lactose
Inactive
Lack of inducer: the lac repressor block all
but a very low level of trans-cription of
lacZYA .
Lactose is present, the low basal level
of permease allows its uptake, andβ-
galactosidase catalyzes the conversion of
some lactose to allolactose.
Allolactoseacts as an
inducer,binding to the lac repressor
and inactivate it.
Response to lactose

37. Response to glucose
The LAC operon

38. Catabolite Repression
 Feed back control of lac operon through Catabolite repression
 Transcription of lac operon takes place with the help of another protein
named catabolite activator protein (CAP for short).
 When a small molecule called cyclic AMP (cAMP) binds CAP it is able to
bind the promoter region of the lac operon
 In the absence of cAMP, CAP fails to bind to the promoter region and hence
no transcription takes place.
 cAMP is produced by an enzyme called adenylcyclase
 In the presence of glucose in the environment the following changes take
place-
o Synthesis of adenylcyclase is inhibited
o cAMP production drops down
o (cAMP – CAP) complex does not form
o CAP fails to bind to the promoter sequence
o Transcription of lac operon does not take place
 Acts like a feed back mechanism
o Lactose ------- Beta-galactosidase -----> Glucose ↑ + Galactosidase