Protein Synthesis

The Genetic Code

DNA Þ RNA (4 nt) Þ Protein (20 a.a.)

4¹ = 4 a.a.; 4² = 16 a.a.; 4³ = 64 a.a. Þ therefore, 3 nt to code for one amino acid!!

Triplet: all code words are composed of 3 sequence nucleotides

Way to figure out genetic code: poly U = UUUUU = poly Phe, therefore UUU = Phe
Start = AUG (met) = used for initiation and elongation; Stop = UAA, UAG, UGA

Degeneracy: since there are 64 code words and only 20 a.a., some a.a. are specified by more than one triplet

Get mutation in 3^rd position Þ possible to have no effect at all

Non-overlapping: triplets are read sequentially; No part of one code word is used in a preceding or subsequent code word Þ yields a reading frame

If we add one nt Þ will shift the reading frame Þ frameshift mutation Þ changes entirety of protein

Universiality: same genetic code is used in all organisms with few exceptions (mitochondria)

Major Components Involved in Protein Synthesis

mRNA template

prok: multiple coding regions can be contained on a single mRNA (polycistronic = many genes)
euk: mRNAs encode only a single open reading frame (monocistronic = one gene)

aminoacyl tRNAs

cloverleaf

no proof reading involved

individual tRNAs specific for each triplet in the genetic code

~ 50 tRNAs

tRNA synthetases recognize which a.a. goes to which specific tRNA and covalently attaches it to the 3' end

20 a.a. thus ~20 tRNA synthetases

some add the a.a. to the 2'OH and some to 3'OH Þ CCA

synthetases recognize different tRNAs via recognition and discriminatory elements

must be highly specific because no way to proofread after this point (thus 2^nd genetic code)

activation of tRNA (aminoacylation)

a.a. + ATP Þ a.a.~AMP + PPi Þ (+ tRNA)Þ a.a.~tRNA + AMP

the high energy bond of ATP is preserved in the ester linkage of a.a.~tRNA; this energy will later be used in peptide bond formation

not all tRNAs are alike

the initiator tRNA binds in a different place than elongation tRNA, thus 2 types of met tRNA
in prok: block the end with a formyl group = fmet: blocks terminus so they can’t be used for elongation

Ribosome

2 subunits: small subunit (prok 30s/ euk 40s); large subunit (prok 50s/ euk 60s)

subunit contains proteins (50 – 80) and rRNA (3 – 4) (60% of mass)

It’s the RNA that does the work

Small subunit + initiator + large subunit

Provides site of assembly of components and a peptidyl transferase center (catalyzes peptide bond formation)

Translates message from 5' Þ 3': assembles protein N Þ C orientation

One messenger maybe translated by several ribosomes at a time: polyribosomes or polysomes; spaced ~150 nt apart; the nascent proteins begin to fold up into tertiary conformation before synthesis is complete

Mechanics of Protein Synthesis

Initiation

In Prokaryote:

Race btwn synthesizing RNA, synthesizing protein, and degradation of RNA
To control regulation: have multiple genes under one promoter (operon); or give all the genes the same operon and hope they all initiate at the same time (polycistronic mRNA)
initiator genes – Shine Delgarno sequence: purine rich (A and G) = consensus seq; starting sites have different signal strengths; signal recognized by base pairing with small ribosomal subunit

In Eukaryote:

CAP recognition factor binds which causes small subunit to bind
the small subunit scans mRNA and binds to 1^st AUG it meets
Thus eukaryote have CAP to know where to start while prokaryote have internal initiaion
If poly A tail present, will initiate much faster; used to discourage translation of incomplete mRNAs
When small subunit binds to mRNA, it already has met bound to it
The interaction of this tRNA with the 1^st AUG signals the binding of the large subunit (this fixes the reading frame)

Elongation

P site = peptidyl; A site = amino acyl

Initiator binds to P site and all others bind to A site

Ribosome cycle

P site filled with initiator or tRNA~polypeptide Þ the next aminoacyl tRNA binds in A site (requires elongation fators and GTP)

The polypeptide chain in P site attacks a.a. in A site Þ catalyzed by peptidyl transferase ctr (part of 50s subunit)

Peptide bond synthesis generates peptidyl tRNA in A site Þ translocation of ribosome, moves pep~tRNA into P site (requires factors and GTP)

After translocation, ribosome is restored to beginning state

Termination

No tRNAs can recognize the "stop" codons
When ribosome reaches the stop codon, release factors bind to the ribosome and release it from the mRNA

Protein Synthesis Inhibitors

Streptomycin Þ inhibits initiation in prokaryotes

Tetracycline Þ inhibits binding of aminoacyl tRNA to the A site in prokaryotes

Chloramphenicol (prok), cyclohexamide (euk) Þ inhibits peptide bond formation

Puromycin Þ inhibits prokaryote and eukaryote translocation