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