protein metabolism the genetic code protein synthesis (translation) protein targeting &...
TRANSCRIPT
Protein Metabolism
• The genetic code
• Protein synthesis (translation)
• Protein targeting & degradation
What are needed for protein synthesis?
>70 ribosomal proteins>20 enzymes to activate amino acid precursors>12 proteins/enzymes for the initiation, elongation & termination of polypeptides~100 enzymes for the final processing of proteins>40 tRNAs & rRNAs
The most complex biosynthetic process!
Three major advances for the understanding of protein synthesis
1) Paul Zamecnik et al., 1950s (p.1021)newly synthesized, radiolabeled proteins are accumulatedat small ribonucleoprotein particle (i.e., ribosome) in liver.
Three major advances for the understanding of protein synthesis (cont’d)
2) Mahlon Hoagland & Zamecnikactivated amino acids are attached to a heat-stable soluble RNA(i.e., tRNA), forming aminoacyl-tRNAs. Aminoacyl-tRNA synthetases are involved.
3) Francis Crick’s adaptor hypothesisa small nucleic acid (perhaps RNA) could serve the role of an adaptor, one part binding a specific a.a. and aother part recognizing the nt sequence encoding that a.a. in the mRNA.
The triplet, nonoverlapping code
Insertion or deletion mutations alter the sequence of triplets.Adding/subtracting 3 nt leaves the remaining triplet intact,providing evidence that a codon has 3 nt.
codon: a triplet of nucleotides that codes for a specific amino acid.reading frame: established by the first codon, then begins a new one every 3 nucleotide residues.
The Genetic Code Was Cracked Using Artificial mRNA Templates
Marshall Nirenberg, 1961enzymatic methods to synthesize
poly(U) > phenylalanine poly(C) > proline poly(A) > lysine …
Nirenberg & Philip Leder, 1964trinucleotides induce specific binding of aminoacyl-tRNA to ribosome.
H. Gobind Khorana, 1960schemical methods to synthesize polynucleotides with repeating sequences of 3 & 4 bases > polypeptides
“Dictionary” of amino acid code words as they occur in mRNAs.
termination codon (red)
initiation codon (green)
Each reading frame gives a different sequence of codons,but only one is likely to encode a given protein.
Open reading frame (ORF): a reading frame without a termination codon among 50 or more codons.
Protein Metabolism
• The genetic code
• Protein synthesis (translation)
• Protein targeting & degradation
“Dictionary” of amino acid code words as they occur in mRNAs.
termination codon (red)
initiation codon (green)
The Ribosome Is a Complex Supramolecular Machine
Masayasu Nomura et al., 1960s (p.1037)both ribosomal subunits can be broken down into theirRNA and protein components, then reconstituted in vitro.
Ribosomal subunits are identified by their S (Svedberg unit)values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge.
Models for the secondary structure of E. coli 16S and 5S rRNAs
The sequences of the rRNAs of many organisms have been determined. Each has a specific three-dimensional conformation featuring extensive intrachain base pairing.
Transfer RNAs Have Characteristic Structure Features
Robert H. Holley et al., 1965 (p.1038)yeast tRNAAla
cloverleaf conformation 苜蓿葉形
Protein Synthesis
• Stage1: Aminoacyl-tRNA synthetases attach the correct amino acids to their tRNAs
• Stage 2: A specific amino acid initiates protein synthesis
• Stage 3: Peptide bonds are formed in the elongation stage
Proofreading by aminoacyl-tRNA synthetase
e.g., Ile-tRNAIle synthetase favors activation of Ile over Valby a factor of 200, i.e., it distinguishes between Val and Ile.
Interaction between an aminoacyl-tRNA synthetase and a tRNA: a “second genetic code”
Recognition sites by:unique enzyme (orange)several enzymes (green)all enzymes (blue)
The tRNAAla elements recognized by the Ala-tRNA synthetase are usually simple.Just a single G=U base pair (red)!
Synthetic simpleform also works!
Protein Synthesis
• Stage1: Aminoacyl-tRNA synthetases attach the correct amino acids to their tRNAs
• Stage 2: A specific amino acid initiates protein synthesis
• Stage 3: Peptide bonds are formed in the elongation stage
Met + tRNAfMet + ATPMet-tRNAfMet + AMP + PPi
N10-Formyltetrahydrofolate + met-tRNAfMet
tetrahydrofolate + fMet-tRNAfMet
transformylase
synthetase
tRNA
The distinction between initiating AUG and internal oneis straightforward...
Three steps of initiation:
Aminoacyl sitePeptidyl siteInitiation Factor (IF)
the initiation complex forms in the expense of the hydolysis of GTP to form GDP and Pi.
Protein Synthesis
• Stage1: Aminoacyl-tRNA synthetases attach the correct amino acids to their tRNAs
• Stage 2: A specific amino acid initiates protein synthesis
• Stage 3: Peptide bonds are formed in the elongation stage
Peptide Bonds Are Formed in the Elongation Stage
Elongation step 1: Binding of the second aminoacyl-tRNA
Elongation requires:• the initiation complex• aminoacyl-tRNAs• elongation factor (EF-Tu, -Ts,-G)• GTP
Proofreading on the ribosome:EF-Tu.GTP/EF-Tu.GDP complexes (~milliseconds) provide opportunitiesfor the codon-anticodon interactions.
How to know GTP is involved?
GTP analogslows hydrolysis, improving the fidelity (by increasing the proofreading intervals) but reducing the rate of protein synthesis.
Energy cost of fidelity in protein synthesis
More than 4 high-energy bonds are required for the formation of each peptide bond of a polypeptide:
2 ATP/GTP during aminoacyl-tRNA formation2 GTP during the first elongation step & translocation
Rapid translation of a single mRNAby polysome in both prok./euk. cells
Polysome:a fiber between adjacent ribosomesin the cluster of 10 to 100.
Protein Synthesis Is Inhibited by Many Antibiotics and Toxins
e.g., disruption of peptide bond formation by puromycin
Peptidyl puromycin
Protein Metabolism
• The genetic code
• Protein synthesis (translation)
• Protein targeting & degradation
Signal sequences of some eukaryotic proteins:
George Palade...David Sabatini & Gunter Blobel, 1970
Signal recognition particle (SRP)
Glycosylation Plays a Key Role in Protein Targeting
Synthesis of the core oligosaccharide of glycoproteins
Tunicamycin mimics the structure ofUDP-N-acetylglucosamine (UDP-GlcNAc) and blocks the first step of glycosylation.
Cells Import Proteins by Receptor-Mediated Endocytosis
coated pit
clathrin
Joe Goldstein& Mike Brown
Protein Degradation Is Mediated by Specialized Systems in All Cells
Proteasome: (Mr 1 x 106)The ATP-dependent, ubiquitin involved proteolytic system in eukaryotes.
Three-step cascade pathway by which ubiquitin is attached to a protein.
The Nobel Prize in Chemistry 2004
"for the discovery of ubiquitin-mediated protein degradation"
Technion – Israel Institute of Technology Haifa, Israel b. 1947
Technion – Israel Institute of Technology Haifa, Israel b. 1937(in Karcag, Hungary)
University of California Irvine, CA, USA b. 1926
AaronCiechanove
AvramHershko
Irwin Rose