• There are many steps between genotype and phenotype; genes cannot by themselves produce a phenotype.

• The expression of a gene takes place in two steps:

– Transcription makes a single-stranded RNA copy of a segment of the DNA.

– Translation uses information encoded in the RNA to make a polypeptide.

DNA, RNA, and the Flow of Information

Mov. translation

The Genetic Code

• mRNA 상의염기서열➡단백질의아미노산서열

• mRNA is read in three-base segments called codons.

• The number of different codons possible is 64 (43),because each position in the codon can be occupied by one of four different bases.

• The 64 possible codons code for only 20 amino acids and the start and stop signals.

The Genetic Code

• AUG, which codes for methionine, is called the start codon, the initiation signal for translation.

• Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes to end translation.

Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes

• tRNA has three functions:

– It carries an amino acid.

– It associates with mRNA molecules.

– It interacts with ribosomes.

Preparation for Translation: Linking RNAs, Amino Acids, and

Ribosomes

• At the 3’ end of every tRNA moleculeis a site to which its specific amino acid binds covalently.

• Midpoint in the sequence are three bases called the anticodon.

• The anticodon is the contact pointbetween the tRNA and the mRNA.

• The codon and anticodon unite by complementary base pairing.

Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes

• Amino acids are attached to the correct tRNAs by activating enzymes called aminoacyl-tRNA synthetases.

• The enzyme has a three-part active site that binds:– A specific amino acid– ATP– A specific tRNA, charged with a

high-energy bond

Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes

• Each ribosome has two subunits: a large one and a small one.

• In eukaryotes the large one has three different associated rRNAmolecules and 49 different proteins.

• The small subunit has onerRNA and 33 different proteinmolecules.

• When they are not translating, the two subunits are separate.

Overview of protein synthesis in prokaryote

• 개시, 연장, 종결 단계마다 특이한 인자 필요

• 개시 ➡ IF1, IF2, IF3

• 연장 ➡ EF-Tu, EF-Ts, EF-G

• 종결 ➡ RF1, RF2, RF3

• Ribosomal protein, 전 과정에 관여 ;

각 Factor, 특정의 과정에 관여

• polysome structure

Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes

• The proteins and rRNAs are held together by ionic bonds and hydrophobic forces.

• The large subunit has four binding sites:– The T site where the tRNA first lands – The A site where the tRNA anticodon

binds to the mRNA codon– The P site where the tRNA adds its

amino acid to the polypeptide chain– The E site where the tRNA goes before

leaving the ribosome

Translation: RNA-Directed Polypeptide Synthesis

• Translation begins with an initiation complex: a charged tRNA with its amino acid and a small subunit, both bound to the mRNA.

• This complex is bound to a region upstream of where the actual reading of the mRNA begins.

• The start codon (AUG) designates the first amino acid in all proteins.

• The large subunit then joins the complex.

• The process is directed by proteins called initiation factors.

* Initiation sequences

* Initiation complex

Eukaryotic protein synthesis

• not fMet but Met, no Shine-Dalgarno sequence

• mRNA와 eIF-4E (cap binding), eIF-4G 외개시인

자 (helicase 활성) 관여

* Initiation complex (Eu.)

Mov. translation-1

Translation: RNA-Directed Polypeptide Synthesis

• Ribosomes move in the 5’-to-3’ direction on the mRNA.

• The peptide forms in the N–to–C direction.

• The large subunit catalyzes two reactions:– Breaking the bond between the

tRNA in the P site and its amino acid

– Peptide bond formationbetween this amino acid and the one attached to the tRNA in the A site

• This is called peptidyl transferase activity.

Translation: RNA-Directed Polypeptide Synthesis

• After the first tRNA releases methionine, it dissociates from the ribosome and returns to the cytosol.

• The second tRNA, now bearing a dipeptide, moves to the P site.

• The next charged tRNA enters the open A site.

• The peptide chain is then transferred to the P site.

• These steps are assisted by proteins called elongation factors.

* Elongation (Pro)

Mov. Protein elongation

* Elongation (Pro)

Translation: RNA-Directed Polypeptide Synthesis

• When a stop codon—

UAA, UAG, or UGA—

enters the A site, a release

factor and a water

molecule enter the A site,

instead of an amino acid.

• The newly completed

protein then separates

from the ribosome. http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter15/animations.html#

Mov. Life Protein syn

Regulation of Translation

• Antibiotics are defensive molecules produced by some fungi and bacteria, which often destroy other microbes.

• Some antibiotics work by blocking the synthesis of the bacterial cell walls, others by inhibiting protein synthesis at various points.

• Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are unaffected.

* Ribosomes

*Chloramphenicol은원핵세포의 50S의단백질합성저해,

특히 mitochondrial ribosome에손상을주므로주의하여사용하여야됨

*Diphtheria toxin은 EF-2의 ADP ribosylation을촉매하여진핵세포의

단백질합성을저해한다

*이외단백질합성을저해하는항생제는 Clindamycin, Aminoglycoside

*30S에작용:

Streptomycin: 결핵치료제Neomycin, Kanamycin, Amikacin, Gentamicin, Tobramycin:

대장수술전장내세균억제Spectinomycin: 임질균치료제Tetracycline제제 (doxycycline, minocycline): 콜레라, 쯔쯔가무시병

*50S에작용:

Chloramphenicol: 뇌막염치료제, 장티푸스균Erythromycin: 레지오넬라균Clindamycin: 그람음성세균, 항생제관련대장염Griseofulvin: 곰팡이성질환

Protein synthesis inhibitors

Regulation of Translation

• Polysomes are mRNA molecules with more than one ribosome attached.

• These make protein more rapidly, producing multiple copies of protein simultaneously.

* Polysomes

Proteolytic Cleavage

1. Most proteins →→proteolytic cleavage following translation

2. Many proteins →→synthesized as inactive precursors (Pancreatic enzymes)

3. Inactive precursor proteins →→proproteins

4. A complex example of post-translational processing →→prepro-

opiomelanocortin (POMC) synthesized in the pituitary

Proteolytic Cleavage

5. Another example of a preproprotein is insulin (Next)

6. Inactive precursors →→zymogens

7. Zymogens are activated by proteolytic cleavage →→blood clotting cascade

Primary structure of human proinsulin and insulin.

단백질의 수식과 이동(insulin)

in ER

in processing

in ER (complex protein Conformation: Hsps

in processing

Acylation1. Many proteins →→modified at their N-termini 2. Initiator methionine →→hydrolyzed and an acetyl group →→added to the new N-terminal amino

acid 3. Acetyl-CoA →→acetyl donor

4. 14 carbon myristoyl group →→added to their N-termini5. The donor →→myristoyl-CoA →→membranes protein6. Cyclic AMP-dependent protein kinase (PKA) →→myristoylated

Phosphorylation1. Phosphorylation →→most common protein modifications2. Phosphorylations→→regulate the biological activity of a protein: Next page3. Phosphate →→added and later removed4. The enzymes that phosphorylate proteins →→kinases5. Remove phosphates →→phosphatases6. Protein kinases: ATP + protein <----> phosphoprotein + ADP7. Serine, threonine and tyrosine →→phosphorylation8. The ratio of phosphorylation →→1000/100/1 for serine/threonine/tyrosine9. Although the level of tyrosine phosphorylation is minor10. Importance of phosphorylation of this amino acid →→numerous growth factor receptors is controlled by tyrosine phosphorylation

PhosphorylationPhosphorylations →→regulate the biological activity of a protein

Sulfation 1. Sulfate modification of proteins occurs at tyrosine residues such as in fibrinogen 2. The universal sulfate donor →→3'-phosphoadenosyl-5'-phosphosulphate (PAPS)3. Sulfate →→permanently

Figure 12.16 Posttranslational Modifications to Proteins