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Protein Metabolism
Protein Synthesis
Protein Synthesis: an overview
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PS proceeds from N -terminus to Cterminus (amino to carboxyl)
Ribosomes:read mRNA in 5’ to 3’
direction
Polyribosomes (polysomes)
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Max. density ~ 1 ribosome/80 nucleotides
Protein Synthesis: an overview
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Chain elongation: links the growing chain to
incoming tRNA’s AA residue
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AA residues: added to C-terminus
Ribosome (prokaryote) has 3 tRNA binding sites
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The P site: peptidyl site
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Binds peptidyl –tRNA
Peptidyl-tRNA holds polypeptide
After peptide bond formation, binds deacylated tRNA
The A site: amino-acyl site
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Binds incoming aminoacyl-tRNA
Protein Synthesis: an overview
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Ribosome (prokaryote) has 3 tRNA binding sites
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After peptide bond formation
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Deacylated tRNA released from P site
Replaced in P site by newly formed peptidyl-tRNA
A site is vacated
The E site: exit site
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Recent finding
Largely confined to 50S subunit
Deacylated tRNA dissociate from the ribosome
Protein Synthesis: an overview
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Five (5) major stages of protein synthesis
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1.Activation of Amino Acids
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20 AA
20 aminoacyl-tRNA synthetases
20 or more tRNAs (min 32)
ATP
Mg2+
Protein Synthesis: an overview
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Five (5) major stages of protein synthesis
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2.Initiation
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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
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Five (5) major stages of protein synthesis
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3.Elongation
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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
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Five (5) major stages of protein
synthesis
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4. Termination and Release
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Termination codon in mRNA
Polypeptide releasing factor: RF1, RF2, RF3
ATP
5. Folding and Processing
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Enzymes (lots!)
Cofactors (lots!)
P S: stage one; activation of AA
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Takes place in the cytosol
Each AA is attached to specific tRNA
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ATP to AMP + Ppi
Catalyzed by MG 2+ -depending aminoacyltRNA synthetases
Aminoacylated tRNA is said to be
charged
P S: stage two; Initiation
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Initiation codon recognized by tRNAfmet
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N-formylmethionine residue (fmet)
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Special tRNA
Special aminoacyl-tRNA synthetase
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First: bind tRNAfmet with Met
Then: N –formulates the Met residue
Note: proteins are post-translationally
modified
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Deformylation of fmet residue
Sometimes: removal of N-terminal Met
P S: stage two; Initiation
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Initiation codon recognized by tRNAfmet
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In eukaryotes:
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All polypeptides synthesized by cytoplasmic
ribosomes
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Begin with Met residues (not
Have special initiating tRNA
met)
f
Mitochondrial/chloroplast products are like
polypeptides
P S: stage two; Initiation
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The Shine-Dalgarno Sequence:
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John Shine and Lynn Dalgarno in 1974
An initiating signal in mRNA
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Base pair with (antiparallel):
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8-13 bp to the 5’ side of initiation codon
4-9 purine residues
Complementary pyrimidine-rich sequence
Nearnear 3’ end of 16SrRNA on 30S subunit
mRNA-rRNA interactions
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Sets mRNA in the correct position
Initiation of transcription
P S: stage two; Initiation
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Three Stages: assemble Initiation
Complex
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Requires Initiation factors
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Not permanently associated with ribosome
3 (in E. coli ): IF-1, IF-2, IF-3
Stage one (1):
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30S subunit binds IF-3, IF-1
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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
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Three Stages: assemble Initiation
Complex
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Stage two (2):
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30S subunit with IF-3 with mRNA binds w/IF-2
IF-2 is already bound to:
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GTP
fMet-tRNA fmet
Anticodon and codon pair
P S: stage two; Initiation
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Three Stages: assemble Initiation
Complex
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Stage three (3):
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Large complex formed in stage 2 combines
with 50S subunit
GTP to GDP and Pi
IF-3, IF-1, IF-2 are released
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IF-3 released before 50S attaches
irreversible
P S: stage two; Initiation
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Three Stages: assemble Initiation
Complex
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At end of Initiation:
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fMet-tRNA fmet with mRNA with Ribosome
complex is formed
fMet-tRNA fmet in P site
A site ready
Eukaryotic Initiation is similar
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More initiation factors
No Shine-Dalgarno
Cap is located; the 1st AUG downstream
P S: stage three; Elongation
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3 stage cycle that is repeated
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Adds AA to C-terminus
Up to 40 residues/sec
Elongation factors (EF)
P S: stage three; Elongation
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Aminoacyl-tRNA binding
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GTP with EF-Tu with aminoacyl-tRNA binds to
ribosome
aminoacyl-tRNA: bound in codon-anticodon
interaction at A site
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GTP to GDP + Pi
EF-Tu to GDP + Pi are released
Regenerate GTP
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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
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Transpeptidation:
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Peptide bond formation
Transfer of N-formylmethionyl group
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From tRNA in P site
To amino group of 2nd AA in A site
Forms a dipeptidyl-tRNA in A site
tRNA fmet in P site
Peptidyl transferases
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Catalyzes bond formation on LG subunit
Catalyzed by 23S rRNA (Harry Noller, 1992)
P S: stage three; Elongation
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Translocation:
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Ribosomes moves toward 3’ end by one
codon
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Shift requires
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Dipeptidyl-tRNA moves to Psite
Deacylated- tRNA fmet released
New codon (3rd) into A site
EF-G (translocase)
GTP
Believed to be accompanied by 3-D
changes in ribosome
P S: stage three; Elongation
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Repeat elongation cycle
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Need 2 GTP for each added AA residue
Protein chain always remains attached to a
tRNA
P S: stage four; TERMINATION
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Signaled by termination codon
When termination codon is in the A site
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3 releasing factors
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RF1, RF2, RF3
Hydrolysis of terminal peptidyl-tRNA bond
Release of protein and last tRNA
Dissociation of ribosome
P S: stage four; TERMINATION
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When termination codon is in the A site
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3 releasing factors
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RF1: reacts to UAG, UAA
RF2: reacts to UGA, UAA
RF3: ?
RF bind at termination codon
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Peptidyl transferase gives chain to H2O
Eukaryotes
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One RF: eRF
Recognizes all 3 termination codons
P S: stage five; post-translational
modification
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To become mature, polypeptides must fold to
native conformations
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Disulfide bonds must form
Multisubunit proteins: subunits must combine
Must be modified by enzymes
Proteolytic cleavage
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Most common P-T modification
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Eg: all proteins have fMet residue removed
Eg: Conversion of trypsinogen to trypsin
P S: stage five; post-translational
modification
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Covalent modifications
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E.g.:
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Methylations
Hydroxylations
Etc
Changes in functional groups and radical
groups