Aris Kaksis Riga University docent - Chemistry

Biochemistry of Medicinals I Phar 6151 CHAPTER SIX
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor

Protein Synthesis: Translation

I.          Ribosome

The synthesis of a polypeptide chain is a complex process that not only involves mRNA and tRNA but is coordinated by molecular complexes containing both rRNA and multiple proteins (ribosomes). Ribosomes catalyze peptide synthesis according to the instructions from mRNA.

A.        Structure

The bacterial ribosome is composed of protein (1 / 3 of total mass) and ribosomal RNA - rRNA (2 / 3 total mass). RNA is critical from ribosomal structure and function. Ribosome is a massive ribo-nucleo-protein assembly with a molecular weight of 2 700 kD and a diameter of 200 Å. The size is also defined by the sedimentation coefficient S which for the ribosome is 70S. The bigger the coefficient, the bigger the entity.
On average there are 20 000 ribosomes per E. coli cell, or 1 / 4 the molecular weight MW.

            Overall Ribosome Structure: Translation Machine

50S Subunit ®

5S RNA
30S Subunit ®

30S Subunit ®

• tRNA site, peptidyl transferase, GTPase and Exit site

II.        Polymerization

Synthesis occurs by the reading mRNA sequence, 5'--->3', and a peptide is made form the amino N-terminus to the ---> carboxyl C-terminus.
DNA        5'-ATG--GCC-TTT--GAT--TCT-AAA--TAA- 3’                  Translating the Message
RNA       5' -AUG--GCC-UUU--GAU--UCU-AAA--UAA--3’
Protein   N- Met    Ala    Phe    Asp    Ser    Lys    Stop -C Overview of Translation site

•           Translation and transcription in bacteria occur in close proximity

A.        Initiation

Protein synthesis in bacteria begins with the codon AUG or methionine, however, the first 1st amino acid AA is
                                                                                                                  actually N-formyl-methionine (fMet ).

•     N-formyl-methionine is attached to a special tRNA, (initiator tRNA or tRNAF). It is synthesized by formylation of Methionine-tRNAF

Start Signal
•     Approximately, 5-10 bases upstream from the AUG sequence is a purine rich sequence called the
                                                             Shine-Delgarno sequence (S.D.).
•     3’ end of the 16S RNA from 30S ribosomal subunit binds to the S.D. by specific base pairing:

                    Shine-Delgarno sequence (S.D.)
16S                                       UCCUCCA—7 6 5 4 3 2 1 —Ñ1
mRNA      5'-GAUUCCU—AGGAGGU—UUGACCU——AUG-GCC-UUU-3'
Protein                                                                                N-fMet    Ala   Phe

• Initiation requires ribosome binding to S. D. sequence and binding of fMet initiator tRNA to the START
                                                                                                                                                 codon (AUG).
• mRNA and tRNAF are brought to the ribosome with the help of initiator factors (IF1, IF2, IF3):
1) 30S subuint forms a complex with all three factors
2) GTP binding to IF2 enables mRNA and fMet-tRNAF to join the complex (IF3 is released)
3) GTP hydrolysis provides the energy DG < 0 evolving for binding 50S subunit and the release of IF1 and IF2
4) 70S complex with mRNA and fMet-tRNAF is called INITIATOR COMPLEX
5) fMet-tRNAF is bound in the P (peptidyl) site of the ribosome, while A (aminoacyl) site is free

B.        Elongation

Elongation requires the escorting of the appropriate AA-tRNAs to the ribosome, peptide bond formation, translocation along ® the mRNA, and proofreading.

• Elongation is facilitated by the transport of amino-acylated-tRNAs by elongation factor Tu (EF-Tu) to
                                                                       the ---> ribosome A site. GTP hydrolysis drives this process.
      Release of GDP to EF-Tu is facilitated by EF-Ts
•     Release of Ts by GTP binding to Tu
• EF-Tu does not interact with fMET-tRNAF

1.      Fidelity and Proofreading

The fidelity of protein synthesis depends on having the correct AA in the A site at the time of peptide bond formation. Elongation must have the ability to check if the right amino acid AA is being added, because it cannot be removed once incorporated.

•         Peptide bond is not formed immediately: incorrect aminoacyl-tRNA has the time to leave A site
•           Each aminoacyl-tRNA is scrutinized both before and after GTP hydrolysis in EF-Tu. Correct
aminoacyl-tRNA bind mRNA strongly in both conformationally different states, while           incorrect
aminoacyl-tRNAs dissociate
•         Error rate of 1 in 10 000 (<10-4 Err / b) is generally sufficient for proteins 100-1000 AA in length

2.        Translocation

The movement ® of the ribosome along mRNA, as the polypeptide is being synthesized, requires;

• movement ¬ of the tRNA and polypeptide from the A (AA-tRNA) site to the P (peptide) site to the
                                E (exit) site, which is assisted by the hydrolysis of GTP by translocase (EF-G).
• a change in the interaction of the tRNAs with 50S after peptide bond formation.
• a mechanism that insures that premature termination will be rare by holding on to the peptide-tRNA

           E      P       A                                                         E      P       A

E P A E P A

---> Continued Peptide Synthesis

3.         Termination

Stop codons, UAA, UGA or UAG, are recognized by proteins called release factors (RFs):
•           RF1 recognizes UAA or UAG
•           RF2 recognizes UAA or UGA
•           RFs promote the hydrolysis of the peptide-tRNA ester bond
•           protein leaves first 1st, followed by tRNA and then mRNA
•           70S dissociates into 50S and 30S
•           IF3 binding to 30S prevents premature association with 50S

III.    Drugs that Stop Translation

Antibiotics interfere with the processes of translation by several different mechanisms.

<--------------

<----------------

||-----> Release of uncharged tRNA

<---------------

<--------------------------

Genetic code

	U	C	A	G
U	UUU-Phe UUC-Phe UUA-Leu UUG-Leu	UCU-Ser UCC-Ser UCA-Ser UCG-Ser	UAU-Tyr UAC-Tyr UAA-Term UAG-Term	UGU-Cys UGC-Cys UGA-Term-SelenoCys-Trp-Mitochondria UGG-Trp
C	CUU-Leu CUC-Leu CUA-Leu CUG-Leu	CCU-Pro CCC-Pro CCA-Pro CCG-Pro	CAU-His CAC-His CAA-Gln CAG-Gln	CGU-Arg CGC-Arg CGA-Arg CGG-Arg
A	AUU-Ile AUC-Ile AUA-Ile AUG-Met	ACU-Thr ACC-Thr ACA-Thr ACG-Thr	AAU-Asn AAC-Asn AAA-Lys AAG-Lys	AGU-Ser AGC-Ser AGA-Arg AGG-Arg
G	GU-Val GUC-Val GUA-Val GUG-Val	GCU-Ala GCC-Ala GCA-Ala GCG-Ala	GAU-Asp GAC-Asp GAA-Glu GAG-Glu	GGU-Gly GGC-Gly GGA-Gly GGG-Gly