Translation espyr1 S10 translation

(1)

LSM1401 Summary 10 © Lim Fang Jeng

1

Translation

- Triplet code ( Non-overlapping, no discontinuity) - Known as codon (4³=64) configurations

- Contains Third base degeneracy ( Except Met and Trp)

- Same amino acid can be coded by different codon (The converse is not true)

- The codes are universal, it is almost the same in both eukaryotes and prokaryotes (with a few exceptions) - Degeneracy of base pairs act as a buffer against mutation

o Minimize misreading of codes

tRNA

- Generally L shaped

- One terminal is specifically covalently bonded with an amino acid (specific for each tRNA depends on the anticodon)

- The other terminal consist of three-base anticodon sequence (Antiparallel to codon) - Enzyme involved: aminocyl-tRNA synthetase (add amino acid to tRNA)

Translation (All require ATP/GTP, Mg

²⁺

)

Prokaryotes

Steps Components

Amino acid activation Amino acids tRNAs

aminocyl-tRNA synthetase

Initiation fmet-tRNA^fmet (SPECIALIZED for initiation only) AUG (Start ) codon (associated with fmet-tRNA^fmet) 30S, 50S ribosomal units

Initiation factors (IF-1, IF-2, IF-3) Elongation 70S ribosome

codons of mRNA aminocyl-tRNAs EF-Tu, EF-Ts, and EF-G Termination 70S ribosome

STOP codon (UAA, UAG, UGA) RF-1,RF-2, RF-3

(2)

2 Ribosomal-binding site

- Ribosome will bind to a purine-rich region in mRNA (this implies that the 3 end of the 16S RNA has a pyrimidine rich region)

- The ribosomal-binding site = Shine-Dalgarno sequence (5 GGAGGU 3 ) (for Prokaryotes ONLY) - Approximately -10 region

- Note that there is G-U pair (non-canonical), for more general machinery for rRNA Initiation (Formation of 30S and 70S Initiation complexes)

(1) Formation of 30S initiation complex

a. (IF-1 + IF-3 + 30S subunit) binds to mRNA, IF-2, fmet-tRNA^fmet = 30S initiation complex b. IF-2 delivers the initiator tRNA (Requires GTP) into peptidyl (P) site

(2) Formation of 70S initiation complex

a. Loss of initiation factors will enable the binding of 50S subunit to the 30S initiation factor b. Forming 70S initiation complex

c. The acceptor site accepts START codon Elongation

(1) After initiation, the aminocyl-tRNA binds at the A site on the ribosome

a. EF-Tu + GTP is required (2) EF-Tu is released and regenerated

a. EF-Ts and GTP is required

(3) Peptide bond is formed between the amino acid before and the newly synthesized amino acid

a. Peptide bond is formed from the old one TO the NEW one b. Energy is required!

(4) Uncharged tRNA is released a. peptidyl-tRNA  P site

b. unchardes-tRNAE site released c. EF-G + GTP is required

(5) Cycle continues…

Peptide bond formation

- Nucleophilic attack by α-amino group to Carbonyl C of the P site amino acid - It is facilitated when a lone pair of N-3

of purine moiety (A²⁴⁵¹) abstracts the proton

- This process happens in ribosome Termination

When STOP codon is found in the mRNA a. Release factor (NOT tRNA!!!!!!!!)

b. RF-1 (UAA and UAG) or RF-2 (UAA and UGA) (One of the two) c. RF-3 does not bind to any codon, but facilitates the RF-1 or RF-2

d. Blocks new tRNA to bind to the mRNA and hydrolyses the carboxyl end of the peptidyl tRNA e. Whole complex dissociates, it can be reused and proceed to next phase of translation

tRNA binds here

(3)

3

Eukaryotes

- Involves more eukaryotic initiation factors

- initiator tRNA carries only Met and functions only in initiation (tRNAiMet – NOT formylated) - mRNA is post-transcriptional modified (Capping, PolyA tail and splicing)

Initiation

(1) Formation of 43S preinitiation complex

a._Met-tRNAi binds with eIF2 and GTP and move to 40S subunit  43S subunit b. This is known as 43S preinitiation complex

(2) Formation of 48S preinitiation complex

a. Kozak sequence form complex with 43S complex (mRNA is added here) b. Scan for AUG (START) codon

(3) Formation of 80S initiation complex

a. 60S subunit + 48S = 80S initiation complex b. Energy is required

(4) Translation begins…

Elongation and Termination - Similar to prokaryotes

- It forms a loop structure due to the polyA binding protein - binds to eIF4G in eukaryotes

- Elongation involves polyribosomes (not only one as in prokaryotes!!!) - One transcript can code for many polypeptides!

- eEF1A =EF-Tu ; eEF1B=EF-Ts; eEF2= EF-G - In eukaryotes, ONLY one release factor eRF1 Inhibitors in Translation

- Often used as antibiotics in medical field

- Often we are advised to finish the antibiotics because it will have a potential to cause microbes(bacteria) to mutate and then it will be resistant to the antibiotics, causes the antibiotics does not function

anymore.

Post-Translational Modification

- Folding of peptide chain to form protein - Driven by non-covalent bond formation - Most frequent: glycosylation

- Often occurs in endoplasmic reticulum and being transported to Golgi Apparatus - Example: Preproinsulin  Insulin

Examples of posttranslational peptide cleavage

(4)

4 There are 3 pathways for protein folding

(1) Chaperone-independent folding: Protein folds as it is synthesized (2) Hsp70 assisted protein folding : Hsp70 binds to polypeptide

chains and assist the folding

(3) Assisted by Hsp70 and cheperonin complexes: GroEL and GroES in prokaryotes (E.coli) ; TRiC /CCT in eukaryotes

Most proteins fold by (1) or (2).