Il dogma centrale della Biologia DNA trascrizione inversa trascrizione 5’ 3’ RNA 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 è letto a triplette Analisi dei mutanti nel locus rII del fago T4 (F. Crick and S. Brenner, 1961). Ceppo T4 Ceppo 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. wt CAU CAU CAU CAU CAU Insersione CAU ACA UCA UCA UCA Delezione CAU ACA UCA UCA UCA G 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 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.

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) 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

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

Leu, Arg and Ser have 3 different tRNAs Arg Met 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 Leu Pro Arg Gln Asn Ser Ile Thr Leu, Arg and Ser have 3 different tRNAs Lys Arg Met Asp Val Ala Gly Glu Wobbling occurs because the conformation of the tRNA anticodon loop permits flexibility at the first base of the anticodon. codon anticodon

Modifications occur in all parts of the tRNA molecule 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 Anticodon UAI -NH2 According to wobbling rules a single tRNA can not recognize four codons -NH2

Changes of codon meaning in Mammaliam Mitochondria 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)