Structural basis of biological function

• Prokaryotes - In cytoplasm
• Eukaryotes - In cytoplasm or on the ER membrane.

Components

• tRNA - Clover leaf shape
•             70 nucleotides long
•              Amino acid joins to 3' end (CCA) at 2' or 3' hydroxyl of the A
•              Contains modified bases dihydrouridine (D), ribothyamine (T), pseudouridine (psi),                     inosine (I), or methylguanosine (mG). 
•              tRNA can undergo wobble base pairing
•                         5' - 3' anticodon - codon: I -> C, A, U
•                                                                 U - A, G
•                                                                 G - C, U
•                                                                  A -> U
•                                                                  C-> G

• ribosome
•            Has a large and a small subunit (50 & 30 in prokaryotes or 60 & 40 in eukaryotes).
•             Eukaryotes = 80S
•              Prokaryotes - 70S
• Three sites: Aminoacyl - entry site for charged tRNA
•                     peptidyl - site of peptide bond formation
•                     exit - site of exit for tRNA

• aminoacyl-tRNA synthetase binds to ATP, creating AMP, which forms an amino acid - AMP complex.
• tRNA bonds, releasing the AMP and creating a high energy ester bond.
• It is a nucleophilic attack

• IF1 binds to the A site, and IF3 binds to the small subunit. 
• 16s rRNA binds to the Shine Dalgarno sequence. 
• IF2-GTP-formyl-methionine complex with a tRNA binds to the small subunit, and the anticodon pairs with the AUG on the mRNA at the P site.
• IF3 and IF1 dissociate, allowing the large/small subunit to connect and freeing the A site (respectively),
• GTP hydrolysis releases IF2. 

1. eIF1 and eIF1A bind to the A site.

2. eIF3 binds to the small subunit.

3. The eIF2- methionine complex is still there, but without the GTP.

4. The GTP is attached to eIF5.

All of this is the 43S preinitiation complex

5. mRNA is attaches to eIF4A, B, E(binds 5' cap), and G (binds the polyA tail using polyA binding proteins PABPC). 

eIF3 attaches to eIF4G, connecting the mRNA complex and 43S complex, forming the 48S preinitiation complex.

6. The polyA tail and 5' cap dissociate and eIF2 looks for the kozac sequence

7. eIF5B guides the large subunit into position and binds to eIF1A.

8. eIF5 GTP hydrolysis by eIF2 releases everything bar the 5b/1A complex.

9. The GTP hydrolysis of eIF5B releases the rest of the complex, and the polyA tail and 5' cap reassociate.

1. EF-TU-GTP delivers the aminoacyltRNA to the A site. 

2. Correct anticodon/codon base pairing causes GTP hydrolysis, releasing EF-TU.

3. A peptide bond forms by peptidyl transferase activity using the 23S rRNA, a ribozyme.

                     This is a condensation reaction, and so releasing H2O.

4. EF-G-GTP binds to the small subunit, and GTP hydrolysis causes the ribosome to slide along the mRNA, moving the contents of the P site to the E site, and the A site to the P site. 

5. The E site tRNA is ejected, and the process repeats. 

the EF-TU is replaced by eEF1 - alpha

The EF-G is replaced by eEF2

The 23S ribozyme is replaced by a 28S ribozyme.

1. 2 release factors bind to stop codons in the A site:

                   RF1 - UAA and UAG

                   RF2 - UAA and UGA

2. RF3 detects when RF1 and RF2 have interacted correctly.

3. Upon correct interaaction, RF3 hydrolyses GTP and RF1 or 2 cleaves the peptidyl-tRNA bond.

• RF1/2 is replaced by eRF1
  
• RF3 is replaced by eRF3.