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.

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

tRNA (green)

EF-G

GDP (red)

C-terminus (green)mimics tRNA

X174

Overlapping Genes in Different Reading FramesAre Found in Some Viral DNAs

Overlapping Genes in Different Reading FramesAre 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 theirRNA 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.

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

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

Mg2+

Aminoacyl-tRNA synthetase

Amino acid + tRNA + ATP

aminoacyl-tRNA + AMP + PPi

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)

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 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

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

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.

The initiating AUG is guided by the Shine-Dalgarno sequencein 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 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.

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 analogslows hydrolysis, improving the fidelity (by increasing the proofreading intervals) but reducing the rate of protein synthesis.

Termination of polypeptide synthesisrequires 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 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.

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

e.g., addition of isoprenyl groups

Posttranslational modification (cont’d)

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)

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 ofUDP-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

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.

Three-dimensional structure of the eukaryotic proteasome

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