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Il dogma centrale della Biologia
DNA
trascrizione
trascrizione
inversa
5’
3’ RNA
traduzione
N-terminus
C-terminus
proteina
Il codice genetico
41= 4 possibilità
42=16 possibilità
43=64 possibilità
20 aminoacidi
The sequence of a coding strand
of DNA, read in the direction
from 5’ to 3’, consists of
nucleotide triplets (codons)
corresponding to the amino acid
sequence of a protein read from
N-terminus to C-terminus.
Il codice genetico è letto a triplette
Analisi dei mutanti nel locus rII del fago T4 (F. Crick and S. Brenner, 1961).
Ceppo T4
rIIrII+
Ceppo E. coli
B
grandi, rotonde
piccole, irregolari
K
no placche
piccole, irregolari
Induzione del fenotipo rII- mediante l’uso di
proflavina che provocava inserzioni o
delezioni di singole coppie di basi.
A
(studio della ricombinazione genica, Benzer, 1950)
Studiando una particolare mutazione indotta dalla
proflavina, denominata FCO, Crick e collaboratori
trovarono delle reversioni rivelate dalla
formazione di placche normali su cellule del ceppo
di E. coli K.
G
L’analisi genetica rivelò che i revertanti non erano
identici al wild type. La retromutazione non era
un’esatta inversione della mutazione originale ed
era dovuta ad una seconda mutazione in un sito
diverso.
wt
CAU CAU CAU CAU CAU
Insersione
CAU ACA UCA UCA UCA
Delezione
CAU ACA UCA UCA UCA
CAU ACA UAU CAU CAU
Sistemi di sintesi proteica cell-free (M. Nirenberg, 1961)
Incorporazione di aminoacidi radioattivi nelle proteine in un sistema in vitro
La prima grande conquista fu la scoperta del procedimento per
ottenere mRNA sintetico. Se si mescolano i ribonucleotidi ad
elevata conc. con l’enzima polinucleotide fosforilasi la reazione
produce RNA a singolo filamento (la sintesi non richiede il DNA
stampo e i nucleotidi sono incorporati a caso).
La Decifrazione del Codice Genetico
1) Poli (U) mRNA  polifenilalanina
2) Poli (A) mRNA  polilisina
3) Poli (C) mRNA  poliprolina
4) Poli (G) mRNA  no traduzione (H-DNA)
5) Polimeri casuali di A e C in cui:
[A]>[C]  polipeptide composto da Asn e Gln (2A+C)
[C]>[A]  polipeptide composto da His e Thr (2C+A)
6) Poli (CA)  polipeptide risultante: Thr-His-Thr-His
7) Poli (UC)  polipeptide risultante: Leu-ser-Leu-Ser
Nel 1963 Nirenberg e Matthaei avevano identificato
l’aminoacido associato con 40 codoni (composizione ma
non l’ordine dei nucleotidi)
Ribosome binding assay (Nirenberg and
Leder, 1965)
+
+
X-X-X
Trinucleotide
da saggiare
(seq. nota)
Filtro di nitrocellulosa
Solo il complesso ribosomaaminoacil-tRNA
(corrispondende al codone in esame)trinucleodite
è trattenuto sul filtro mentre gli aminoacil-tRNA
liberi non vengono trattenuti
The fMet-tRNA is used only for initiation. It
recognizes AUG (Met) or GUG (Val) and
occasionally UUG (Leu). The extent of initiation
declines about half when AUG is replaced by
GUG and by about half yet again when UUG is
employed.
61 codons represent amino acids
and 3 cause termination (TERM)
The reduced specificity at the last position is
known as third-base degeneracy.
Often one tRNA can recognize more than
one codon. This means that the base in the
first position of the anticodon must be able
to partner alternative bases in the
corresponding third position of the codon.
Impiego del codice genetico e dei codoni in E. coli e nell’uomo
The number of codons for each amino
acid does not correlate with the frequency
of use in proteins
L’ RNA transfer
74-95
nucleotidi
There are 20
aminoacyl-tRNA
synthetase, each
recognizing a single
amino acid and all the
tRNAs onto which it
can legitimately be
placed.
Mutation
amino acid
CUC  CUG
no effect (Leu)
= pseudouridina
CCU  AUU
Leu  Ile
UUU  UCU
Phe  Tyr
UUC  UAC
Phe  Tyr
mG e m2G= metil e
dimetilguanosina
mI= metilinosina
DHU o UH2=
diidrouridina
A group of isoaccepting tRNAs must be charged
only by the single aminoacyl-tRNA synthetase
specific for their amino acid. There are 20
aminoacyl-tRNA synthetase in the cell.
Once a tRNA has been charged, the
amino acid plays no further role in its
specificity, which is determined
exclusively by the anticodon
Nonsense mutations suppression
(intergenic suppression)
There are 3 separate genes coding for tRNATyr 
Frameshift mutations suppression
(intragenic suppression)

Stop codon
Base mutata
Phe
Tyr
Ser
Leu
Cys
 stop 
Often one tRNA can recognize more than one
codon, codon-anticodon recognition involves
wobbling.
There are only three cases in which a unique
meaning is conferred by the presence of a particular
base at the third position: AUG (for methionine),
UGG (for tryptophan), and UGA (termination).
Trp
His
Leu
Ile
Gln
Asn
Ser
Lys
Arg
Thr
Met
Val
Arg
Pro
Ala
Leu, Arg and Ser have 3 different tRNAs
Asp
Gly
Glu
codon
anticodon
Wobbling occurs because the
conformation of the tRNA
anticodon loop permits flexibility
at the first base of the anticodon.
Modifications occur in all parts of the tRNA molecule.
There are more than 50 different types of modified
bases in tRNA (D, T and  were originally transcribed as
U while mG, dmG, I and mI were transcribed as G).
When bases in the anticodon are modified,
further pairing patterns become possible in
addition to those predicted by the regular and
wobble pairing involving A, C, U, and G.
Ile codons
AUA
AUU
AUC
-NH2
-NH2
Anticodon UAI
According
to wobbling
rules a
single tRNA
can not
recognize
four codons
Some modifications create preferential readings of
some codons
1) 2-thiouracil pairs only with A
2) Anticodons with uridine-5-oxyacetic acid and 5methoxyuridine in the first position recognize A and G
efficiently as third bases of the codon, but recognize U
less efficiently
3) Queuosine and its derivatives (modified G bases)
continue to recognize both C and U, but pair with U
more readily.
Changes of codon meaning in Mammaliam Mitochondria
Stop
(UC)
Ile
(UC)
Arg
(UC)
There are sporadic alterations of the
universal code
The universality of the genetic code is
striking, but some exceptions exist. They
tend to affect the codons involved in
initiation or termination
Exceptions to the universal genetic code also
occur in the mitochondria from several
species (they have been evolved separately).
UC = Universal Code, the anticodon is in parenthesis