Protein Metabolism

Protein Synthesis

Protein Synthesis: an overview

PS proceeds from N -terminus to C-terminus (amino to carboxyl)

Ribosomes:read mRNA in 5’ to 3’ direction

Polyribosomes (polysomes) Max. density ~ 1 ribosome/80

nucleotides

Protein Synthesis: an overview Chain elongation: links the growing

chain to incoming tRNA’s AA residue AA residues: added to C-terminus Ribosome (prokaryote) has 3 tRNA binding

sites The P site: peptidyl site

Binds peptidyl –tRNA Peptidyl-tRNA holds polypeptide After peptide bond formation, binds deacylated

tRNA The A site: amino-acyl site

Binds incoming aminoacyl-tRNA

Protein Synthesis: an overview

Ribosome (prokaryote) has 3 tRNA binding sites

After peptide bond formation Deacylated tRNA released from P site Replaced in P site by newly formed peptidyl-tRNA A site is vacated

The E site: exit site Recent finding Largely confined to 50S subunit Deacylated tRNA dissociate from the ribosome

Protein Synthesis: an overview

Five (5) major stages of protein synthesis 1.Activation of Amino Acids

20 AA 20 aminoacyl-tRNA synthetases 20 or more tRNAs (min 32) ATP Mg2+

Protein Synthesis: an overview

Five (5) major stages of protein synthesis 2.Initiation

mRNA N-Formylmethionyl-tRNA Initiation codon in mRNA (AUG) Ribosome: 30S and 50S subunit Initiation factors: IF-1. IF-2, IF-3 GTP Mg2+

Protein Synthesis: an overview

Five (5) major stages of protein synthesis 3.Elongation

Initiation complex (functional 70S ribosome) aminoacyl-tRNAs specified by codons Peptidyl transferases Elongation factors: EF-Tu. EF-Ts, EF-G GTP Mg2+

Protein Synthesis: an overview Five (5) major stages of protein

synthesis 4. Termination and Release

Termination codon in mRNA Polypeptide releasing factor: RF1, RF2, RF3

ATP 5. Folding and Processing

Enzymes (lots!) Cofactors (lots!)

P S: stage one; activation of AA

Takes place in the cytosol Each AA is attached to specific

tRNA ATP to AMP + Ppi

Catalyzed by MG 2+ -depending aminoacyl-tRNA synthetases

Aminoacylated tRNA is said to be charged

P S: stage two; Initiation

Initiation codon recognized by tRNAfmet

N-formylmethionine residue (fmet)

Special tRNA Special aminoacyl-tRNA synthetase

First: bind tRNAfmet with Met

Then: N –formulates the Met residue

Note: proteins are post-translationally modified

Deformylation of fmet residue Sometimes: removal of N-terminal Met

P S: stage two; Initiation

Initiation codon recognized by tRNAf

met

In eukaryotes: All polypeptides synthesized by

cytoplasmic ribosomes Begin with Met residues (not f

met) Have special initiating tRNA

Mitochondrial/chloroplast products are like polypeptides

P S: stage two; Initiation The Shine-Dalgarno Sequence:

John Shine and Lynn Dalgarno in 1974 An initiating signal in mRNA

8-13 bp to the 5’ side of initiation codon 4-9 purine residues

Base pair with (antiparallel): Complementary pyrimidine-rich sequence Nearnear 3’ end of 16SrRNA on 30S

subunit mRNA-rRNA interactions

Sets mRNA in the correct position Initiation of transcription

P S: stage two; Initiation Three Stages: assemble Initiation

Complex Requires Initiation factors

Not permanently associated with ribosome 3 (in E. coli ): IF-1, IF-2, IF-3

Stage one (1): 30S subunit binds IF-3, IF-1

Prevents premature joining of LG and SM subunits mRNA binds to 30S

Initiation codon (AUG) to P-site on 30S subunit Guided by Shine-Delgarno sequence

P S: stage two; Initiation Three Stages: assemble Initiation

Complex Stage two (2):

30S subunit with IF-3 with mRNA binds w/IF-2

IF-2 is already bound to: GTP fMet-tRNA fmet

Anticodon and codon pair

P S: stage two; Initiation Three Stages: assemble Initiation

Complex Stage three (3):

Large complex formed in stage 2 combines with 50S subunit

GTP to GDP and Pi

IF-3, IF-1, IF-2 are released IF-3 released before 50S attaches

irreversible

P S: stage two; Initiation Three Stages: assemble Initiation

Complex At end of Initiation:

fMet-tRNA fmet with mRNA with Ribosome

complex is formed fMet-tRNA f

met in P site A site ready

Eukaryotic Initiation is similar More initiation factors No Shine-Dalgarno Cap is located; the 1st AUG downstream

P S: stage three; Elongation

3 stage cycle that is repeated Adds AA to C-terminus Up to 40 residues/sec Elongation factors (EF)

P S: stage three; Elongation Aminoacyl-tRNA binding

GTP with EF-Tu with aminoacyl-tRNA binds to ribosome

aminoacyl-tRNA: bound in codon-anticodon interaction at A site

GTP to GDP + Pi

EF-Tu to GDP + Pi are released Regenerate GTP

EF-Tu with GDP + EF-T3 to EF-Tu with EF-Ts + GDP

EF-Tu with EF-Ts + GTP to EF-Tu with GTP + EF-Ts

P S: stage three; Elongation Transpeptidation:

Peptide bond formation Transfer of N-formylmethionyl group

From tRNA in P site To amino group of 2nd AA in A site

Forms a dipeptidyl-tRNA in A site tRNA f

met in P site Peptidyl transferases

Catalyzes bond formation on LG subunit Catalyzed by 23S rRNA (Harry Noller,

1992)

P S: stage three; Elongation Translocation:

Ribosomes moves toward 3’ end by one codon

Dipeptidyl-tRNA moves to Psite Deacylated- tRNA f

met released New codon (3rd) into A site

Shift requires EF-G (translocase) GTP

Believed to be accompanied by 3-D changes in ribosome

P S: stage three; Elongation Repeat elongation cycle

Need 2 GTP for each added AA residue

Protein chain always remains attached to a tRNA

P S: stage four; TERMINATION

Signaled by termination codon When termination codon is in the A

site 3 releasing factors

RF1, RF2, RF3 Hydrolysis of terminal peptidyl-tRNA bond Release of protein and last tRNA Dissociation of ribosome

P S: stage four; TERMINATION

When termination codon is in the A site 3 releasing factors

RF1: reacts to UAG, UAA RF2: reacts to UGA, UAA RF3: ?

RF bind at termination codon Peptidyl transferase gives chain to H2O Eukaryotes

One RF: eRF Recognizes all 3 termination codons

P S: stage five; post-translational modification To become mature, polypeptides must

fold to native conformations Disulfide bonds must form Multisubunit proteins: subunits must combine Must be modified by enzymes

Proteolytic cleavage Most common P-T modification

Eg: all proteins have fMet residue removed Eg: Conversion of trypsinogen to trypsin

P S: stage five; post-translational modification

Covalent modifications E.g.:

Methylations Hydroxylations Etc

Changes in functional groups and radical groups