Jul 16, 2015
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).
Processing and Folding
Post-translational modification
Post-translational modification
Proteolytic cleavage
Modification of aminoacids
Subunit aggregation
Protein folding and chaperones
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
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
Proteolytic cleavage
Proteolyticcleavage
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).
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.
Proteolytic cleavage
Modification of amino acids
Modification of amino acids
Modification of aminoacids
Modification of amino acids
SUBUNIT AGGREGATION
PROTEIN FOLDING AND CHAPERONES
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.
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.
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
►Gene Expression is Controlled by
Regulation Proteins: Activators and
Repressors1.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.
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.
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.
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.
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
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.
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
Lactose operon: a regulatory gene and 3 stuctural genes, and 2 control elements
lacI
Regulatory gene
lacZ lacY lacA DNA
m-RNA
β -GalactosidasePermease
Transacetylase
Protein
Structural GenesCis-acting elements
PlacI PlacOlac
The LAC operon
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
i p o z y a
Very low level of lac mRNA
Absence of lactose
Active
i p o z y a
b-Galactosidase
PermeaseTransacetylase
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.
Allolactose acts as an inducer, binding to the lac repressor
and inactivate it.
Response to lactose
Response to glucose
The LAC operon
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 inhibitedo cAMP production drops downo (cAMP – CAP) complex does not formo CAP fails to bind to the promoter sequenceo Transcription of lac operon does not take place
Acts like a feed back mechanismo Lactose ------- Beta-galactosidase -----> Glucose ↑ + Galactosidase