Tre linee di ricerca hanno

portato alla scoperta che il

DNA è il materiale ereditario

Il principio trasformante (Griffith, 1928)

Figure. The general structure of

nucleotides. Left: computer model.

Right: a simplified representation.

Figure. The chemical structure of

pentose which contains five carbon

atoms, labeled as C1' to C5'. The

pentose is called ribose in RNA and

deoxyribose in DNA, because the

DNA's pentose lacks an oxygen

atom at C2'. Recalling that RNA

stands for "ribonucleic acid", and

DNA for "deoxyribonucleic acid".

Figure. Formation of the phosphodiester bond through

the condensation reaction.

Like peptide chains, a nucleic acid chain also has

orientation: its 5' end contains a free phosphate group

and 3' end contains a free hydroxyl group. Synthesis

of a nucleic acid chain always proceeds from 5' to

3'. Therefore, unless specified otherwise, the sequence

of a nucleic acid chain is written from 5' to 3' (left to

right).

Figure. A nucleic acid chain. Its 5' end

contains a free phosphate group. The 3'

end has a free hydroxyl group.

In DNA or RNA, a nucleic acid chain is also called a

strand. A DNA molecule typically contains two strands

whereas most RNA molecules contain a single strand.

The length of a nucleic acid chain is represented by the

number of bases. In the case of a double-stranded

nucleic acid, bases are paired between two

strands. Therefore, its length is given by the number of

base pairs (bp). 1 kb = 1000 bases or bp; 1 Mb = 1

million bases or bp. Oligonucleotides refer to short

nucleic acid chains (< 50 bases or bp) and

polynucleotides have longer chains.

The function of RNA

polymerases

Both RNA and DNA

polymerases can add

nucleotides to an existing

strand, extending its

length. However, there

is a major difference

between the two classes

of enzymes: RNA

polymerases can initiate

a new strand but DNA

polymerases

cannot. Therefore,

during DNA replication,

an oligonucleotide

(called primer) should

first be synthesized by a

different enzyme.

Figure. The chemical

reaction catalyzed by

RNA polymerases.

Figure. Computer model of base

pairing in DNA. In a normal DNA

molecule, adenine (A) is paired

with thymine (T), guanine (G) is

paired with cytosine (C). The

uracil (U) of RNA can also pair

with adenine (A), since U differs

from T by only a methyl group

located on the other side of

hydrogen bonding.

A DNA molecule has two

strands, held together by the

hydrogen bonding between

their bases.

As shown in the above figure, adenine can form two hydrogen bonds with thymine; cytosine

can form three hydrogen bonds with guanine. Although other base pairs [e.g., (G:T) and

(C:T) ] may also form hydrogen bonds, their strengths are not as strong as (C:G) and (A:T)

found in natural DNA molecules.

Schematic drawing of DNA's two

strands.

Due to the specific base pairing,

DNA's two strands are

complementary to each

other. Hence, the nucleotide

sequence of one strand determines

the sequence of another strand. For

example, in Figure 3-B-2, the

sequence of the two strands can be

written as

5' -ACT- 3'

3' -TGA- 5'

Note that they obey the (A:T) and

(C:G) pairing rule. If we know the

sequence of one strand, we can

deduce the sequence of another

strand. For this reason, a DNA

database needs to store only the

sequence of one strand. By

convention, the sequence in a DNA

database refers to the sequence of

the 5' to 3' strand (left to right).

DNA polymerases can extend

nucleic acid strands only in the 5'

to 3' direction. However, in the

direction of a growing fork, only

one strand is from 5' to 3'. This

strand (the leading strand) can be

synthesized continuously. The

other strand (the lagging strand),

whose 5' to 3' direction is

opposite to the movement of a

growing fork, should be

synthesized discontinuously.

Figure.

(a) Comparison between the leading strand

and the lagging strand. (b) The primase first

synthesizes a new primer which is about 10

nucleotides in length. The distance between

two primers is about 1000-2000 nucleotides

in bacteria, and about 100-200 nucleotides in

eukaryotic cells. (c) DNA polymerase

elongates the new primer in the 5' to 3'

direction until it reaches the 5' end of a

neighboring primer. The newly synthesized

DNA is called an Okazaki fragment. (d) In

E. coli, DNA polymerase I has the 5' to 3'

exonuclease activity, which is used to

remove a primer. (e) DNA ligase joins

adjacent Okazaki fragments.

The whole lagging strand is synthesized by

repeating steps (b) to (e).

In a DNA molecule, the two strands are not

parallel, but intertwined with each other. Each

strand looks like a helix. The two strands

form a "double helix" structure, which was

first discovered by James D. Watson and

Francis Crick in 1953. In this structure, also

known as the B form, the helix makes a turn

every 3.4 nm, and the distance between two

neighboring base pairs is 0.34 nm. Hence,

there are about 10 pairs per turn. The

intertwined strands make two grooves of

different widths, referred to as the major

groove and the minor groove, which may

facilitate binding with specific proteins.

Figure. The normal right-handed "double

helix" structure of DNA, also known as the B

form.

In a solution with higher salt concentrations or with alcohol added, the DNA structure may

change to an A form, which is still right-handed, but every 2.3 nm makes a turn and there are

11 base pairs per turn.

Another DNA structure is called the Z form, because its bases seem to zigzag. Z

DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. The DNA

molecule with alternating G-C sequences in alcohol or high salt solution tends to

have such structure.

Figure. Comparison between B form and Z form.

Le proteine variano ampiamente in grandezza forma e funzione

Organismo Numero di

coppie di

basi

Lunghezza del

DNA (mm)

Dimesioni

dello spazio

cellulare (mm)

Numero di

cromosomi

Batteriofago 4.85 x 104 0,017 < 0,0001 1

Batterio

(Escherichia coli)

4,7 x 106 1,4 0,001 1

Lievito

(Saccharomyces

cervisiae)

1,25 x 107 4,6 0,005 16 (x 1 o 2)

Moscerino della

frutta (Drosophila

melanogaster)

1,65 x 108 56,0 0,010 4 (x 2)

Esseri umani

(Homo sapiens)

3 x 109 999,0 0,010 23 (x 2)

Il contenuto di DNA di varie specie

Organizzazione dei genomi a DNA

Genoma Forma Dimensioni (kb)

Eucarioti ds lineare da 104 a 106

Batteri ds circolare 103

Plasmidi ds circolare (alcuni ds lineari) 2-15

Virus a DNA dei

mammiferi

ss lineare, ds lineare, ds circolare 3-280

Batteriofagi ss circolare, ds lineare 50

DNA dei cloroplasti ds circolare 120-160

DNA mitocondriale ds circolare (alcuni ds lineari) Animali: 16,5

Piante: 100-2500

Genes

By definition, a gene includes the entire nucleic acid sequence necessary for the expression of its product (peptide or

RNA). Such sequence may be divided into regulatory region and transcriptional region. The regulatory region could

be near or far from the transcriptional region. In eucaryotic cells, the transcriptional region consists of exons and

introns. Exons encode a peptide or functional RNA. Introns will be removed after transcription.

As shown in the following figure, a typical DNA molecule consists of genes, pseudogenes and extragenic

region. Pseudogenes are nonfunctional genes. They often originate from mutation of duplicated genes. Because

duplicated genes have many copies, the organism can still survive even if a couple of them become nonfunctional

Figure. General organization of the DNA sequence. Only the exons encode a functional

peptide or RNA. The coding region accounts for about 3% of the total DNA in a human cell.

Duplicated Genes

Most proteins do not need duplicated genes, because the mRNA molecule transcribed from one gene can

be translated into many copies of its protein product. However, rRNA and tRNA are the final gene

products. In order to accelerate the production process, all species contain an array of tandemly repeated

RNA genes. The number of repeats ranges from tens to 24,000.

Number of RNA genes

*The X chromosome of fruit fly contains 250 copies of Pre-rRNAs, Y chromosome contains 150 copies.

There are four types of rRNA in mammalian cells: 28S, 5.8S, 5S and 18S. In the human genome, 28S, 5.8S and 18S are

clustered together. They form a single transcription unit which will be separated by specific enzymes after transcription. "

Pre-rRNA" refers to their precursor. In humans, a repeat unit for the pre-rRNA has about 40 kb in length, including a 13-

kb transcription unit and a 27-kb untranscribed spacer region. The transcription unit contains three spacers: ETS, ITS1 and

ITS2. They will be removed during RNA processing.

b globin gene

Figure. Graphic view of the b

globin gene, which consists of

three exons and two introns, with

a total length of 1.6 kb. This

figure was obtained from NCBI.

Gene family

"Gene family" refers to a set of genes with homologous sequences. For example, H2A, H2B,

H3 and H4 are in the same histone gene family. Their products have similar structures and

functions. Another example is the b-globin gene family located on the chromosome 11.

Figure. The b-

globin gene

family includes

b, d, Ag, Gg

and e. Y is a

pseudogene. H

S1 to HS4 are

regulatory

elements.

Caratteristiche delle sequenze genomiche degli eucarioti

E’ possibile distinguere la frequenza di ripetizione di sequenze

genomiche dalle cinetiche di riassociazione del DNA di un genoma

denaturato.

Dalle cinetiche di riassociazione si individuano due tipi di

sequenze genomiche:

Il DNA non ripetitivo consiste di sequenze uniche di cui ce

ne è una sola copia per genoma aploide.

Il DNA ripetitivo consiste di sequenze presenti in più di una

copia per genoma.

•Le proteine sono in genere codificate da sequenze di DNA

non ripetute.

Soltanto lo 0,1% del genoma umano differisce da una persona

all’altra. Ad eccezione della regione codificante gli antigeni

leucocitari umani (HLA) la variazione genetica è modesta nel

DNA codificante.

Meno del 40% del genoma umano è costituito da geni e da

sequenze correlate a geni.

Il DNA intergenico consiste di: 1) sequenze uniche od in basso

numero di copie; 2) sequenze moderatamente od altamente

ripetitive.

Le sequenze moderatamente od altamente ripetitive si possono

suddividere in due classi principali: (1) elementi sparsi; (2)

sequenze ripetute in tandem.

• Il DNA ripetitivo può essere suddiviso in due categorie

generali:

DNA moderatamente ripetitivo, costituito da sequenze

relativamente corte ripetute nel genoma in genere da 10 a 1000

volte. Sono sequenze disperse nel genoma.

DNA altamente ripetitivo, consiste di sequenze molto corte

(in genere meno di 100 bp) ripetute molte migliaia di volte nel

genoma e spesso organizzate come lunghe ripetizioni in

tandem.

• Nessuna delle due classi si trova nelle regioni codificanti.

• Nello stesso gruppo tassonomico i genomi più grandi non

contengono più geni, ma solo una maggiore quantità di DNA

ripetitivo.

Le proporzioni delle

diverse componenti

di sequenza variano

nei genomi

eucariotici

Elementi dispersi nel genoma

Sono ripetizioni presenti in tutto il genoma che sono trasposoni

(elementi instabili del DNA che si possono spostare in parti

diverse del genoma) o meglio copie degenerate di trasposoni.

Le ripetizioni non sono raggruppate, ma sono sparse in numerose

posizioni all’interno del genoma. Possono essere suddivisi in due

categorie in base alla loro lunghezza:

Sequenze più corte di 500 bp - SINE (short interspersed

nuclear elements); elementi Alu (SINE attivi nell’uomo).

Sequenze più lunghe di 500 bp – LINE (long interspersed

nuclear elements); elementi L1 (LINE attivi nell’uomo).

Classi di elementi trasponibili

Classe Intermedio di trasposizione Esempi

Classe I

Retrotrasposoni LTR RNA Lievito: elementi Ty;

Esseri umani: Retrovirus endogeni

umani (HERV);

Topo: particella A intracisternali

(AP).

Retrotrasposoni non LTR

LINE (autonomi)

SINE (non autonomi)

RNA Esseri umani:

Elementi L1

Elementi Alu

Classe II

Trasposoni di DNA DNA Batteri:

Sequenze di inserzione

Batteriofago Mu

Trasposoni (batterifago Tn7).

Drosophila:

Elementi P.

Mais:

Elementi Ac e Ds.

Invertebrati e vertebrati:

Superfamiglia Tc1/mariner

ITR: ripetizioni terminali invertite; DR: brevi ripetizioni dirette; ORF: modulo di

lettura aperto; LTR, lunghe ripetizioni terminali; HERV, retrovirus endogeni umani;

gag, antigene gruppo specifico; prt, proteasi; Pol, polimerasi; env, involucro; RT,

trascriptasi inversa; EN, endonucleasi; TSD, duplicazioni del sito di bersaglio; UTR,

regione terminale non trascritta.

Sequenze ripetute in tandem

• Le ripetizioni in tandem costituiscono approssimativamente il 10% del

genoma e si dividono in tre classi in base alla lunghezza:

Satelliti: sono costituiti da DNA altamente ripetitivo con una lunghezza

di ripetizione che va da una a parecchie migliaia di coppie di basi.

Queste sequenze sono organizzate in grandi gruppi nelle regioni di

eterocromatina dei cromosomi, vicino ai centromeri ed ai telomeri, e

sono abbondanti anche nel cromosoma Y.

Minisatelliti: loci di ripetizioni in tandem a numero variabile (VNTR),

sono composti da motivi di sequenza che vanno da circa 15 a 50 bp. La

lunghezza totale delle ripetizioni in tandem va da 500 bp a 20 kb.

Microsatelliti o brevi ripetizioni in tandem (STR): l’unità ripetuta va da

2 a 6 bp per una lunghezza totale che varia fra 50 e 500 bp. Le

sequenze STR più comuni sono ripetizioni dinucleotidiche.

• La variazione genetica da individuo ad individuo nei

minisatelliti e STR (polimorfismi) è dovuta soprattutto al

numero di elementi ripetitivi disposti in tandem, ma ci possono

essere piccole differenze anche nella sequenza.

• Queste regioni variabili sono particolarmente utili per la

genetica legale perché si possono usare per generare un profilo

del DNA di un individuo, pur non dando alcuna informazione

sui tratti fenotipici dello stesso.

Chromatin is the substance

which becomes visible

chromosomes during cell

division. Its basic unit is

nucleosome, composed of

146 bp DNA and eight

histone proteins. The

structure of chromatin is

dynamically changing, at least

in part, depending on the need

of transcription . In the

metaphase of cell division,

the chromatin is condensed

into the visible

chromosome. At other times,

the chromatin is less

condensed, with some regions

in a "Beads-On-a-String"

conformation.

Figure. The condensed structure of chromatin.

(a) The 30 nm chromatin fiber is associated with scaffold proteins (notably

topoisomerase II) to form loops. Each loop contains about 75 kb

DNA. Scaffold proteins are attached to DNA at specific regions called scaffold

attachment regions (SARs), which are rich in adenine and thymine.

(b) The chromatin fiber and associated scaffold proteins coil into a helical

structure which may be observed as a chromosome. G bands are rich in A-T

nucleotide pairs while R bands are rich in G-C nucleotide pairs.

A chromosome contains five types of histones: H1 (or H5),

H2A, H2B, H3 and H4. H1 and its homologous protein H5 are

involved in higher-order structures. The other four types of

histones associate with DNA to form nucleosomes. H1 (or H5)

has about 220 residues. Other types of histones are smaller,

each consisting of 100-150 residues.

Figure. Each nucleosome consists of 146 bp

DNA and 8 histones: two copies for each of

H2A, H2B, H3 and H4. The DNA is wrapped

around the histone core, making nearly two turns

per nucleosome.

Figure. The sequence of

H4 from a cow. Lysine

residues (red color) at the

N terminus play a major

role in the regulation of

gene transcription.

An important feature about histones is that they contain a few lysine (K) residues at the N

terminus. Under normal cellular conditions, the R group of lysine is positively charged,

which can interact with the negatively charged phosphates in DNA. The positive R group of

lysine may be neutralized by acetylation, reducing the binding force between histones and

DNA. Such mechanism has been demonstrated to play a major role in the regulation of gene

transcription.

Istone acetiltransferasi (HAT); istone metiltransferasi (HMT); istone chinasi;

istone deacetilasi (HDAC); istone demetilasi; istone fosfatasi.

Atomic Force Microscopy

of Chromatin Fiber

Most cellular RNA molecules are single stranded. They may form secondary structures such

as stem-loop and hairpin.

mRNA is transcribed from DNA, carrying information for protein synthesis. Three

consecutive nucleotides in mRNA encode an amino acid or a stop signal for protein

synthesis. The trinucleotide is know as a codon

Figure. The sequence relationship of DNA, mRNA and the encoded peptide . The sequence

of mRNA is complementary to DNA's template strand, and thus the same as DNA's coding

strand, except that T is replaced by U.

Figure. The

secondary

structure of

tRNA. Blue

color indicates

modified

nucleotides,

with "m"

representing

"methylated". A

nticodon is the

trinucleotides

complementary

to a codon on

mRNA.

The tertiary structure of tRNA. PDB ID = 1TN2

Struttura terziaria del RNA

• I grandi RNA sono composti da domini strutturali.

• Dispositivi per il ripiegamento del RNA: legami ad idrogeno

ed impilamento delle basi.

• I domini preformati con struttura secondaria del RNA

interagiscono per formare la struttura terziaria.

• Interazione del RNA con proteine basiche ed attacco di ioni

metallici mono e/o bivalenti per neutralizzare le cariche

negative del RNA.

• Motivi più comuni: pseudonodo, motivo ad A-minore,

tetranse, cerniere lampo di ribosio, pieghe K.

Motivo a pseudonodo

Motivo A-minore (rRNA)

Motivo a tetraansa

Motivo a piega k

Ripiegamento del

RNA mediato da

proteine

Versatilità della funzione dell’RNA

• Interazione tra molecole di RNA e con DNA a singolo filamento.

• Associazione con proteine, con formazione di complessi RNA-proteine

particelle ribonucleoproteiche od RNP.

• RNA come “impalcatura” particella di riconoscimento del segnale

(SRP).

• RNA della RNP influenza l’attività catalitica della proteina

telomerasi.

• RNA catalitico ribozimi.

• Piccoli RNA che controllano direttamente l’espressione genica

miRNA.

• RNA come materiale ereditario genomi dei virus ad RNA.

In prokaryotes, the

ribosomal RNA (rRNA)

has three types: 23S, 5S,

and 16S. In mammals,

four types of rRNA have

been found : 28S, 5.8S, 5S

and 18S. After rRNA

molecules are produced in

the nucleus, they are

transported to the

cytoplasm, where they

combine with tens of

specific proteins to form a

ribosome. In prokaryotes,

the size of a ribosome is

70S, consisting of two

subunits: 50S and

30S. The size of a

mammalian ribosome is

80S, comprising a 60S and

a 40S subunit. Proteins in

the larger subunit are

designated as L1, L2, L3,

etc. (L = large). In the

smaller subunit, proteins

are denoted by S1, S2, S3,

etc.

During protein synthesis, the

ribosome binds to mRNA and

tRNA as shown in the following

figure. Only the tRNA

containing the anticodon which

matches mRNA's codon may

join the complex.

The mRNA-ribosome-tRNA complex formed

during protein synthesis.

Figure. The standard genetic code. Synthesis of a peptide always starts from methionine (Met), coded

by AUG. The stop codon (UAA, UAG or UGA) signals the end of a peptide. This table applies to

mRNA sequences. For DNA, U (uracil) should be replaced by T (thymine). In a DNA molecule, the

sequence from an initiating codon (ATG) to a stop codon (TAA, TAG or TGA) is called an open reading

frame (ORF), which is likely (but not always) to encode a protein or polypeptide.

Figure. An approach used by Marshall Nirenberg and his colleagues to crack the genetic code.

(i) Synthesize a trinucleotide (e.g. UUU) which mimics a codon in mRNA.

(ii) Prepare various types of aminoacyl-tRNA, e.g., Thr-tRNA, Phe-tRNA, Lys-tRNA, etc.

(iii) Radioactively label an aminoacyl-tRNA (e.g. Phe-tRNA) which might contain the anticodon for

the synthesized trinucleotide.

(iv) Place the trinucleotide, aminoacyl-tRNA and ribosome on a nitrocellulose filter.

Individual trinucleotide and aminoacyl-

tRNA can pass through the filter, but the

ribosome is too big to pass

through. Therefore, if the labeled

aminoacyl-tRNA contains the anticodon

for the trinucleotide, it will bind to the

trinucleotide and ribosome on the

filter. In this case, the radioactivity can

be detected on the filter and the amino

acid in the labeled aminoacyl-tRNA is

likely to be encoded by the

trinucleotide. If no radioactivity was

detected, the trinucleotide is unlikely to

be the codon of the amino acid. Most of

the 64 possible codons can be determined

by repeating this procedure for different

trinucleotides and labellings.

The genetic code is not randomly assigned. If an amino acid is coded by several codons, they often

share the same sequence in the first two positions and differ in the third position. Such assignment is

accomplished by the design of wobble position, but "the evolutionary dynamic that shaped the code

remains a mystery".

Translation is a process by which the nucleotide sequence of mRNA is converted

Translation is carried out by tRNA through the relationship between its anticodon and the associated

amino acid. When a tRNA is brought to the ribosome by the pairing between its anticodon and the

mRNA's codon, the amino acid attached at its 3' end will be added to the growing peptide. In bacteria,

there are 30-40 tRNAs with different anticodons. In animal and plant cells, about 50 different tRNAs are

found. However, there are 61 codons coded for amino acids. Suppose each codon can pair with only a

unique anticodon, then 61 tRNAs would be needed.

Figure. Pairing between tRNA's anticodon and mRNA's codon. The left figure defines the wobble

position where base pairing does not obey the standard rule. The right tables show all possible base

pairings at the wobble position. For example, guanine (G) can pair with both cytosine (C) and uracil

(U) ; inosine (I) can pair with cytosine, adenine and uracil.

In most cases, frameshift involves the insertion or deletion of a single nucleotide in

mRNA. Theoretically, it could involve more than one nucleotide, as long as the number is

not a multiple of 3. When a nucleotide is added to or deleted from the mRNA, the

subsequent sequence will produce an entirely different peptide.

Figure. Illustration of the frameshift. mRNA(a) and mRNA(b) differ by only one

nucleotide: mRNA(b) has an additional nucleotide "G" at the third position in this

figure. Note that the translated amino acids are entirely different after the insertion point.

Wobble pairing

The standard genetic code applies to most, but not all, cases. Exceptions have been found

in the mitochondrial DNA of many organisms and in the nuclear DNA of a few lower

organisms. Some examples are given in the following table.