Il dogma centrale della Biologia
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Il dogma centrale della Biologia. DNA. trascrizione inversa. trascrizione. 5’. RNA. 3’. traduzione. proteina. N-terminus. C-terminus. Il codice genetico.

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Il codice genetico

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Il codice genetico

Il dogma centrale della Biologia

DNA

trascrizione

inversa

trascrizione

5’

RNA

3’

traduzione

proteina

N-terminus

C-terminus

Il codice genetico

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.

41= 4 possibilità

42=16 possibilità

43=64 possibilità

20 aminoacidi


Il codice genetico

Il codice genetico è letto a triplette

Analisi dei mutanti nel locus rII del fago T4 (F. Crick and S. Brenner, 1961).

Ceppo T4Ceppo E. coli

B K

rII- grandi, rotonde no placche

rII+ piccole, irregolari piccole, irregolari

(studio della ricombinazione genica, Benzer, 1950)

Induzione del fenotipo rII- mediante l’uso di proflavina che provocava inserzioni o delezioni di singole coppie di basi.

A

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.

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.

wtCAU CAU CAU CAU CAU

InsersioneCAU ACA UCAUCA UCA

Delezione CAU ACA UCAUCA UCA

G

CAU ACA UAU CAUCAU


Il codice genetico

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).


Il codice genetico

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)


Il codice genetico

Ribosome binding assay (Nirenberg and Leder, 1965)

+

+

X-X-X

Trinucleotide

da saggiare

(seq. nota)

Filtro di nitrocellulosa

61 codons represent amino acids and 3 cause termination (TERM)

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 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.

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.


Il codice genetico

Impiego del codice genetico e dei codoni in E. coli e nell’uomo


Il codice genetico

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)

CCU  AUU Leu  Ile

UUU UCU Phe Tyr

UUC UAC Phe Tyr

  • = pseudouridina

    mG e m2G= metil e dimetilguanosina

    mI= metilinosina

    DHU o UH2= diidrouridina


Il codice genetico

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


Il codice genetico

Nonsense mutations suppression

(intergenic suppression)

There are 3 separate genes coding for tRNATyr 

Frameshift mutations suppression

(intragenic suppression)

Stop codon

Base mutata


Il codice genetico

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).

Phe

Tyr

Cys

Ser

 stop 

Leu

Trp

His

Arg

Pro

Leu

Gln

Asn

Ser

Ile

Thr

Leu, Arg and Ser have 3 different tRNAs

Arg

Lys

Met

Asp

Ala

Gly

Val

Glu

Wobbling occurs because the conformation of the tRNA anticodon loop permits flexibility at the first base of the anticodon.

codon

anticodon


Il codice genetico

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.

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).

Ile codons

AUA

AUU

AUC

Anticodon UAI

-NH2

According to wobbling rules a single tRNA can not recognize four codons

-NH2


Il codice genetico

Some modifications create preferential readings of some codons

1) 2-thiouracil pairs only with A

2) Anticodons with uridine-5-oxyacetic acid and 5-methoxyuridine 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)

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

Ile (UC)

Arg (UC)


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