Protein synthesis decodes the information in messenger RNA

Protein synthesis occurs in three phases:

1. Initiation – the translation machinery locates the start codon in mRNA

2. Elongation – codons are read 5’ 3’ as theprotein is synthesized from the aminoend to the carboxyl end

3. Termination – special proteins hydrolyzethe polypeptide from the last tRNA whena termination codon is reached

Ribosomes have three tRNA-binding sites that bridge the 30S and 50S subunits

The mRNA being translated is bound to 30S

Each tRNA molecule contacts both 30S and 50S

Two of the three tRNAs have anticodon-codoninteractions with the mRNA

A site = aminoacyl siteP site = peptidyl site

The E site (exit site) contains the third tRNA

tRNA acceptor stems are positioned in 50S

The start signal is AUG or GUG preceded by several bases that pair with 16S rRNA

Nearly half the amino terminal aa residues in proteins from E. coli are methionine, suggesting that AUG is the start codon

Initiator regions contain a purine-rich sequencecalled the Shine-Dalgarno sequence about10 nucleotides 5’ of the initiator codon

The Shine-Dalgarno sequence interacts with a complementary region on the 3’ end of 16S RNA

Bacterial protein synthesis is initiated by formylmethionyl (fmet) tRNA

The initiator tRNA (tRNAf) differs from tRNAm used for internal methionine residues

The initiator fmet is removed from about half of the proteins found in E. coli from the newlysynthesized protein

A single aminoacyl-tRNA synthetase links metto both tRNA molecules, however met attached to tRNAf is recognized by a specific enzyme that formylates the met amino group

fMet-tRNAf is placed in the P site during formation of the 70S initiation complex

Initiation factors IF1 and IF3 join the 30S subunit to preventing 30S from prematurely binding 50S

IF2, a GTPase, binds GTP to change shape and enable binding of IF2 to fMet-tRNAf.

The IF2-GTP- fMet-tRNAf complex binds mRNAbound to 16S rRNA via theShine-Dalgarno sequence to create the 30S initiation complex

Binding of 50S causes GTP hydrolysis, IF’s are released and the 70S initiation complex is formed

Elongation factors deliver aminoacyl-tRNA to the ribosome

The mRNA codon in the A site defines which aminoacyl-tRNA will enter the site

Elongation Factor Tu (EF-Tu) delivers the correctaminoacyl-tRNA to the A site when GTP is bound

EF-Tu serves two functions:

1. EF-Tu protects the ester linkage in aminoacyl- tRNA from hydrolysis

2. The GTP in EF-Tu is hydrolyzed to GDP only when an appropriate complex between the EF-Tu-aminoacyl-tRNA complex and the ribosome has formed

EF-Tu is then reset to its GTP form by EF-Ts which induces dissociation of GDP from EF-Tu and replacement by GTP

Peptidyl transferase catalyzes peptide bond formation

When both the A and P sites are occupied by aminoacyl-tRNA, the formylmethionine linked to initiator tRNA is transferred to the amino group in the A site.

The peptidyl transferase center on the 23S subunit of the 50S subunit catalyzes formation of the peptide bond

Formation of a peptide bond is followed by GTP-driven translocation of tRNAs and mRNA

The translocation mechanism

Proteins are synthesized by the successiveaddition of amino acids to the carboxyl terminus

Protein synthesis is terminated by release factors that read stop codons

Stop codons (UAA, UGA or UAG) are read by protein release factors

FR1 recognizes UAA or UAG and RF2 recognizes UAA or UGA

RF3 is a GTPase that mediates interactions between RF1 or RF2 and the ribosome

RF1 and RF2 mimic tRNAs and promote hydrolytic attack on the ester linkage between tRNA and the polypeptide

Prokaryotes and eukaryotes differ in the initiation of protein synthesis

1. Eukaryotic ribosomes are larger, consisting of a 60S large subunit and a 40S small subunit. The 60S subunit contains three RNAs: 5S RNA, 28S RNA and 5.8S RNA. The 40S subunit contains an 18S RNA.

2. The initiating amino acid in eukaryotes is methionine rather than N-formylmethionine. A special initiator tRNA is used called tRNAi

3. The initiating codon is always AUG with no Shine-Dalgarno sequence

Prokaryotes and eukaryotes differ in the initiation of protein synthesis

4. Eukaryotic mRNA is circular. eIF-4E protein binds the 5’ 7-methylguanosine cap and the 3’ poly(A) tail through protein intermediates

Some antibiotics inhibit proteins synthesis

Streptomycin, a highly basic trisaccharide, interferes with binding of formylmethionyl-tRNA to ribosomes preventing initiation

Neomycin, kanamycin and gentamycin interfere with interactions between tRNA and the 16S rRNA to inhibit initiation

Choramphenicol inhibits peptidyl transferase

Erythromycin binds the 50S subunit and blocks translocation

Ribosomes bound to the endoplasmic reticulum manufacture secretory and membrane proteins

Ribosomes attached to the endoplasmic reticulumto form the Rough Endoplasmic Reticulum (RER)

Ribosomes on RER synthesize proteins destined to exit the cell or become membrane proteins exposed on the surface of the cell

Three classes: Secretory proteins; lysosomal proteins; and proteins spanning the plasma membrane

Protein synthesis begins on ribosomes that are free in the cytoplasm

Secretory, lysosomal and membrane proteins begin synthesis on a free ribosome but then arrest until the ribosome binds the cytoplasmic surface of the ER

Docking with the ER restarts protein synthesis with the newly synthesized protein threaded into the lumen of the ER

Signal sequences mark proteins for translocation across the ER membrane

The translocation machinery consists of four components:

1. The Signal Sequence – 9 to 12 hydrophobic amino acid residues, sometimes with positively charged amino acid residues, usually near the amino terminus of the nascent peptide chain. Some signal sequences are maintained in the mature protein while others are cleaved by a signal peptidase.

2. The Signal Recognition Particle (SRP) recognizes and binds the signal peptide then directs the peptide to the ER lumen. SRPs are GTPases.

The translocation machinery consists of four components:

3. The SRP Receptor binds SRP at the surface of the ER membrane. The SRP Receptor is a GTPase.

4. The Translocon is the translocation machinery that transports the nascent polypeptide across the ER membrane.