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Protein metabolism

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					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 accumulated at small ribonucleoprotein particle (i.e., ribosome) in liver.

Three major advances for the understanding of protein synthesis (cont’d)
2) Mahlon Hoagland & Zamecnik activated 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 hypothesis a 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, 1961 enzymatic methods to synthesize poly(U) > phenylalanine poly(C) > proline poly(A) > lysine … Nirenberg & Philip Leder, 1964 trinucleotides induce specific binding of aminoacyl-tRNA to ribosome. H. Gobind Khorana, 1960s chemical 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.

Codon is degenerate: an amino acid may be specified by more than one codon.

Alignment of the two RNAs is antiparallel

Codon pairing relationships when the tRNA anticodon contains inosinate

Protein Metabolism
• The genetic code • Protein synthesis (translation) • Protein targeting & degradation

Wobble Allows Some tRNAs to Recognize More than One Coden

Reading frame and amino acid sequence

“Dictionary” of amino acid code words as they occur in mRNAs.

termination codon (red)

initiation codon (green)

EF-Tu

EF-G

GDP (red)

tRNA (green)

C-terminus (green) mimics tRNA

Overlapping Genes in Different Reading Frames Are Found in Some Viral DNAs

fX174

Overlapping Genes in Different Reading Frames Are Found in Some Viral DNAs: Genes within genes

Protein Synthesis involves five stages

The Ribosome Is a Complex Supramolecular Machine

Masayasu Nomura et al., 1960s (p.1037) both ribosomal subunits can be broken down into their RNA and protein components, then reconstituted in vitro.

Structure of the bacterial ribosome at near-molecular resolution

Ribosomal subunits are identified by their S (Svedberg unit) values, sedimentation coefficients that refer to their rate of sedimentation in a centrifuge.

The sequences of the rRNAs of many organisms have been determined. Each has a specific three-dimensional conformation featuring extensive intrachain base pairing.
Models for the secondary structure of E. coli 16S and 5S rRNAs

Transfer RNAs Have Characteristic Structure Features
Robert H. Holley et al., 1965 (p.1038) yeast tRNAAla

cloverleaf conformation 苜蓿葉形

General cloverleaf secondary structure of all tRNAs

Three-dimensional structure of yeast tRNAPhe deduced from X-ray diffraction analysis

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

Amino acid + tRNA + ATP
Mg2+

Aminoacyl-tRNA synthetase

aminoacyl-tRNA + AMP + PPi

Proofreading by aminoacyl-tRNA synthetase
e.g., Ile-tRNAIle synthetase favors activation of Ile over Val by 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)

Gln-tRNA synthetase

Asp-tRNA synthetase (dimeric)

tRNA (green) bound ATP (red)

The tRNAAla elements recognized by the Ala-tRNA synthetase are usually simple. Just a single G=U base pair (red)! Synthetic simple form 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

Howard Dintzis, 1961 polypeptides grow by addition of new amino acid to the carboxyl end

synthetase

Met + tRNAfMet + ATP Met-tRNAfMet + AMP + PPi

N10-Formyltetrahydrofolate

+ tetrahydrofolate + fMet-tRNAfMet tRNA

met-tRNAfMet

transformylase

The distinction between initiating AUG and internal one is straightforward...

Three steps of initiation:
Aminoacyl site Peptidyl site Initiation Factor (IF) the initiation complex forms in the expense of the hydolysis of GTP to form GDP and Pi.

The initiating AUG is guided by the Shine-Dalgarno sequence in the mRNA

Protein complexes in the formation of a eukaryotic intiation complex

eIF

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 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 opportunities for the codon-anticodon interactions. Elongation step 1: Binding of the second aminoacyl-tRNA

Elongation step 2: Formation of the first peptide bond

Peptide transferase

Elongation step 3: Translocation

EF-Tu/tRNA

EF-G/GDP

How to know GTP is involved? GTP analog
slows hydrolysis, improving the fidelity (by increasing the proofreading intervals) but reducing the rate of protein synthesis.

Termination of polypeptide synthesis requires a special signal Release (or termination) factor

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 formation 2 GTP during the first elongation step & translocation

Rapid translation of a single mRNA by polysome in both prok./euk. cells

Polysome: a fiber between adjacent ribosomes in the cluster of 10 to 100.

A polysome from the silk gland of a silkworm larva

Coupling of transcription and translation in bacteria

Posttranslational modification
e.g., modification of individual amino acids: phosphorylation

e.g., modification of individual amino acids: carboxylation

e.g., modification of individual amino acids: methylation

Posttranslational modification (cont’d)
e.g., addition of isoprenyl groups

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

George Palade... David Sabatini & Gunter Blobel, 1970
Signal sequences of some eukaryotic proteins:

Signal recognition particle (SRP)

Directing eukaryotic proteins with the appropriate signals to the endoplasmic reticulum (ER)

Glycosylation Plays a Key Role in Protein Targeting
Synthesis of the core oligosaccharide of glycoproteins

Tunicamycin mimics the structure of UDP-N-acetylglucosamine (UDP-GlcNAc) and blocks the first step of glycosylation.

Phosphorylation of mannose on lysosome-targeted enzymes, e.g., hydrolase

M-6-P

Proteins Are Targeted to Mitochondria and Chloroplasts by Similar Pathways

Signal Sequences for Nuclear Transport Are Not Cleaved

Bacteria Also Use Signal Sequences for Protein Targeting

Model for protein export in bacteria

Cells Import Proteins by Receptor-Mediated Endocytosis

clathrin

coated pit

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.

Three-dimensional structure of the eukaryotic proteasome

The Nobel Prize in Chemistry 2004

"for the discovery of ubiquitin-mediated protein degradation"

Aaron Ciechanove
Technion – Israel Institute of Technology Haifa, Israel b. 1947

Avram Hershko
Technion – Israel Institute of Technology Haifa, Israel b. 1937 (in Karcag, Hungary)

Irwin Rose
University of California Irvine, CA, USA b. 1926


				
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