Genetic Code, Protein Synthesis Biochem

Genetic Code

There are multiple code words for 1 amino acid. 1 Base coding only has 4 options. 2 Base coding gives 16 options (4×4). 3 base coding gives 64 options (4×4×4) for all 20 amino acids.

mRNA Sequences have Three Possible Reading Frames

• Illustrates point of starting at the same point every time.

• Translation begins NOT at start (5’) of mRNA, but when the AUG code (also code for methionine) is read off.

• It ends when stop codon (UAA, UAG, UGA) is read, not at end of mRNA (3’).

The Standard Genetic Code

• Chemically similar amino acids have similar code words (see Asp, Glu).

  • Green = hydrophobic amino acids

  • Yellow = polar amino acids

  • Red = charged amino acids

• The table has all possible 3 base ACUG code words.

  • Amino acids with 6 code words: Leu, Ser, Arg

  • Amino acids with 1 code word: Met, Trp

  • Amino acids with 4 code words: Most common, similar code words are usually different at the last position.

Representative Examples of Codon Usage in E. Coli and Human Genes

• If an amino acid can have multiple code words, are they all used equally? No,

• Can’t predict code words from trends, see Lys/Glu in E. coli versus humans.

Features of the Genetic Code

Protein Synthesis

• Ribosome binds to mRNA from 5’ end.

• Just 1 rRNA in small subunits, yet still 50-60% of subunit. Similarly with large subunits, ribosomes are very rRNA rich.

• Ribosomes are made of protein complexes and rRNA.

• S values refer to speed at which ribosomes pellet in a centrifuge (sedimentation rate).

The 70S Ribosome of E. coli.

The far right picture has a gap through which mRNA will string, and amino acids will also enter the gap and be linked to the protein chain.

Mechanism of Protein Synthesis

• Protein synthesis has three phases: initiation, elongation and termination.

• Initiation

  • Involves binding of mRNA to the small and large ribosomal subunits, and binding of an initiator tRNA.

• Elongation

  • Includes the formation of all peptide bonds of the growing polypeptide chain (amide bond between carbonyl and amino ends).

• Termination

  • Occurs when a “stop” codon is reached along the mRNA, which leads to the release of the completed polypeptide.

Basic Steps in Protein Synthesis at the Ribosomes

• mRNA is between the 2 subunits of ribosome contacting A, P, E sites.

  • A is acceptor site, P is peptidyl site and E is exit site.

• P site is where protein chain is synthesized, held in place by 3 H-bonds between the codon of an mRNA and the anticodon of the tRNA.

• Protein chain moves back to A site to bind amino acid from the tRNA.

• Sliding ribosome over mRNA, tRNA w/ protein moves from A to P, and another tRNA can be added to A site to begin process anew.

Prokaryotic and Eukaryotic Translation Factors

• Other proteins found in both eukaryotic/prokaryotic cells to facilitates protein synthesis.

• Prokaryotes have 3 initiation, elongation, and release factors each.

• Eukaryotes have at least 12 initiation, 3 elongation and 1 release factors.

  • Eukaryotic elongation factors 1:1 with prokaryotic’s.

Initiation of Protein Synthesis

• mRNA binds such that the first AUG (start code word) binds to the P site.

• IF-1 and IF-3 bind to 30S subunit, IF-2 binds to initiator tRNA bringing in the first methionine.

• GTP breakdown to GDP powers joining of components.

• 50S subunit joins 30s to make 70S initiation complex (also w/ initiator tRNA and mRNA in gap between ribosome subunits)

The Initiator tRNA of E. coli (f-Met-tRNA)

• 2 methionine tRNAs, the regular and the special initiator.

• Initiator tRNA has 3’ end with acceptor stem with methionine covalently linked temporarily.

  • A formyl (aka aldehyde) group bound to the NH (amino group) of methionine, which blocks N-terminal (amino) end such that amino acids can only be added to the carbonyl group.

• 5’-CAU-3’ anticodon of tRNA

• 3’-GUA-5’ mRNA codon

• (aka 5’-AUG-3’ start code word)

Peptide-Chain Elongation

• Binding of a new aminoacyl-tRNA to the A site is brought about by elongation factor EF-Tu. Elongation factor EF-Ts recycles EF-Tu,

• Peptidyl transfer is the step that forms the peptide bond between the new amino acid and the growing polypeptide chain.

  • The peptidyl transferase activity is not due to a protein enzyme, but to an RNA enzyme activity of the “peptidyl transferase center” of the 23S rRNA of the 50S subunit. RNA enzymes are known as ribozymes.

• The process continues with the binding of the next new aminoacyl-tRNA to the A site.

The Elongation Phase

• Methionine rRNA at P site, a new tRNA w/ amino acid wants to come in to A site.

  • The tRNA needs help of activated transition factor EF-Tu, activated by GTP, to slide into A site. A spent GDP is left.

  • EF-Ts removes GDP from EF-Tu and replaces it with GTP to activate it again.

• Peptidyl transfer brings amino acid chain over to A site and elongates the chain.

• Translocation moves ribosome 3 bases to right on mRNA. Needs an activated EF-G (activated by GTP, which is then spent into GDP) to occur.

  • After which, A site is open ot bind another tRNA.

• RATE OF TRANSLATION:

  • Prokaryotes: ~20 aa/sec

  • Eukaryotes: ~8 aa/sec

    • Slow b/c complex process

Termination of Protein Synthesis

• The binding of release factors to the A site transforms the ribosomal peptidyl transferase, that forms the peptide bond between amino acids, into a hydrolase.

• The peptidyl transferase then hydrolyzes the bond linking the polypeptide to its tRNA carrier, thereby releasing the polypeptide.

• Stop codon signifies end of elongation (UAA, UAG, or AGA).

• 23S peptidyl transferase activity changed to hydrolase activity (breaking bonds, adding -H and -OH as ends).

Termination of Protein Synthesis (Visualized)

• RF-1/RF-2 and RF-3 + GTP binding to A site from stop codon, this turns the transferase to hydrolase activity.

• Polypeptide is broken from tRNA carrier.

• Polypeptide released, 70S releases mRNA.

The Ribosome Cycle

• Multiple ribosomes can bind to same mRNA.

• Dynamic equilibrium between 70S and free subunits.

  • Free 30S subunits start initiation all over again.

The Polysome: Multiple Ribosomes Synthesizing Proteins on a Single mRNA Molecule

• This mRNA synthesizes fibroin (β-sheet).

• Arrows point to synthesized proteins.

Protein Folding in the Cell