Nucleosidi

ACETYLTRANSFERASE

SRC-1: different nuclear receptors including other steroid receptors such as GR or ER, and nonsteroid group II

receptors such as VDR,

TIF-2 :PPAR, TR, or RXR, stimulating the transcriptional activity

SRC-3 : signal transducers and activators of transcription (STAT-1) and cAMP response element binding protein

(CREB)

P300/CBP : interacts with a large variety of transcription

factors including AP-1, myoD, Jun, Fos, NF-kB, Pit-1, associates with the p160 family of coactivators; possess

HISTONE ACETYLTRANSFERASE .

PGC1alfa:greatly increases the transcriptional activity of PPARg and the thyroid hormone receptor . PGC-1a also

stimulates mitochondrial .

biogenesis and respiration in muscle cells through regulation of the nuclear respiratory factors (NRFs), which are

transcription factors that regulate genes involved in mitochondrial DNA replication and transcription.

Histone acetylation controls gene expression

Inhibition of histone deacetylation by TSA and TPX (anti-cancer and anti-neurodegenerative drugs)

A strand of DNA winds around histone proteins. When histones are acetylated by a protein called histone

acetyltransferase (HAT), the strand begins to loosen, which allows gene expression.

After a short time, the histones are commonly deacetylated by HDAC, which causes the gene expression to stop.

Trichostatin A (binds to HDAC and inhibits its function. As a result, the histones remain acetylated and gene expression

is activated. Trapoxin works in the same way as trichostatin A.

Coactivators control the physiology of multiple organs that are critical for various metabolic disorders

Co-repressors repress transcription dependent on the presence of certain nuclear receptors.

the NCoR and SMRT corepressors interact with the unliganded heterodimeric partner of specific transcription factors

to recruit histone deacethylase (HDACs) to DNA promoter regions.

The transcriptional repressors operate with three mechanisms by blocking the action of activators.

DNA methylation suppress gene transcription

DNA methylation occurs when cytosine is converted to 5’methyl-cytosine via the actions of DNA methyltransferase

(DNMT). DNA methylation typically occurs at cytosines that are followed by a guanine (i.e., CpG motifs).

CpG islands are unmethylated and are associated with approximately 60-70% of mammalian genes the most in the

promoter region. Methylation is one mechanism for suppressing (or silencing) gene transcription by preventing one or

more transcription factors (TF) and thus RNA polymerase from accessing a gene’s promoter which is required for

transcribing DNA into RNA.

Control of eukaryotic transcription initiation: Ordered binding and function of activators and co-activators result in

cooperative formation of a stable activated initiation complex.

NUCLEAR RECEPTORS: Specific or Metabolic Transcription Factors (Activators-Repressors)

The metabolic nuclear receptors act as metabolic and toxicological

sensors, enabling the organism to quickly adapt to environmental changes by inducing the appropriate metabolic

genes and pathways

Ligands for these metabolic receptors are hormones,compounds from dietary origin, intermediates in metabolic

pathways, drugs, or other environmental factors that, unlike classical nuclear receptor ligands, are present in high

concentrations.

Metabolic receptors are MASTER REGULATORS integrating the homeostatic control of energy and metabolism

processes.

Nuclear Receptors-Transcription factors

•Receptors contain DNA-binding domains and act as ligand-regulated transcriptional activators or suppressors: Effects

of nuclear receptor agonists can persist for hours or days after plasma concentration is zero.

A/B: variable, constitutive activator function AF-1

N-terminal domain variable in sequence/length having activator function (AF-1)

C: conserved DBD

with two C4-zinc fingers which also mediate dimerization

D: hinge

variable hinge-region often carrying an NLS

E: ligand binding domain LBD, ligand-dependent AF-2

conserved larger ligand-binding domain (LBD) functionally complex

ligand-binding, hsp interaction, dimerization, NLS

F: variable C-term

Variable C-terminal domain wihtout specific function

Recettori Nucleari dell’acido retinoico (Tipo II)

L’acido retinoico tutto-trans (RA), si lega a specifici recettori nucleari che appartengono alla famiglia dei recettori

steroidi. Il legame di RA con RAR causa la dimerizzazione con RXR, condizione che inducono modificazioni

conformazionali del recettore (rilascio del corepressore, legame del coattivatore), che determinano l’attivazione di

fattori di trascrizione che portano all’attivazione dei geni correlati agli elementi di risposta RAREs.

RARE= retinoic acid response element

Ruolo dell’acido retinoico (RA):

- Sviluppo embrionale

- Differenziamento dei tessuti epiteliali

- Sviluppo e differenziamento dei linfociti da cui un ruolo nella risposta immunitaria

Key trancription factors (nuclear)

modulated by nutrients (cholesterol,lipids,glucose)

PPARs , peroxisome proliferator-activated receptors

LXR , liver X receptors

SREBPs , sterol-regulatory element-binding proteins

CHREBPs , carbohydrate response element-binding proteins

C/EBP , CCAAT-enhancer-binding protein

FOXO1, forkhead box protein O1

Principali tappe necessarie in una cellule eucariotica

per sintetizzare una proteina dal gene

Ribosomal RNA: rRNA

•Ribosomal RNA is the most abundant form and makes up 80% cellular RNA.

•RNA polymerase I synthesizes rRNA

•Ribosomal RNA molecules are large and are found in the ribososmes.

•Ribosome composition:

o60-65% mass in rRNA

o35-40% mass in protein

•Ribosome subunits:

otwo: a large and a small

Ribosomal RNA synthesis takes place in the nucleolus

RNA Polymerase l

Rrn3-regulated Pol I initiation and cell growth, and indicates a general architecture of eukaryotic transcription initiation

complexes

The RNA polymerase I produces in the nucleolus a precursor rRNA of 45S. Its subsequent processing favored by

small nucleolar ribonucleoprotein particles snoRNP form 5.8S, 18S and 28S r RNA.

Eukaryotic rRNA processing involves small nucleolar ribonucleoproteins

snoRNA are RNA/protein complex. The snoRNA molecule contains an antisense element (10-20 nucleotides), which

are base complementary to the sequence surrounding the base (nucleotide) targeted for modification in the pre-RNA

molecule. This enables the snoRNP to recognise and bind to the target RNA. Once the snoRNP has bound to the

target site, the associated proteins are in the correct physical location to catalyse the chemical modification of the

target base.

Ribosome Types

•In prokaryotes: 23S, 5S,16S

•In eukaryotes: 28S, 5.8S, 5S, 18S

Sintesi del tRNA

tRNA represents 15% of total RNA in the cell

tRNAs are small consisting of 70-90 nucleotides

•there are about 32 different tRNAs in most organisms

•tRNAs function to deliver the amino acids to the ribosomes for protein synthesis

Specific sites in the tRNA

The 5’ – terminal acceptor stem is a 7-bp stem made by the base pairing of the 5'-terminal

nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'-terminal group used

to attach the amino acid). The acceptor stem may contain non-Watson-Crick base pairs.

The CCA tail is a CCA sequence at the 3' end of the tRNA molecule. This sequence is

important for the recognition of tRNA by enzymes critical in translation. In prokaryotes, the

CCA sequence is transcribed. In eukaryotes, the CCA sequence is added during

processing and therefore does not appear in the tRNA gene.

The D arm is a 4 bp stem ending in a loop that often contains dihydrouridine.

The T anticodon arm is a 5 bp stem containing the sequence TΨC where Ψ is a

pseudouridine.

Bases that have been modified, especially by methylation, occur in several positions

outside the anticodon. The first anticodon base is sometimes modified to inosine (derived

from adenine) or pseudouridine (derived from uracil).

tRNA function

When charged by attachment of a specific amino acid to their 3’-end to become

aminoacyl-tRNA , tRNA molecules act as adapter in protein synthesis.

1. Aminoacylation of tRNA

First, the aminoacyl-tRNA synthetase attaches adenosine monophosphate(AMP) to the –

COOH group of the amino acid to creat an aminoacyl adenylate intermediate. Then the

appropriate tRNA displaces the AMP.

2. Aminoacyl-tRNA synthetases

The synthetase enzymes contact their cognate tRNA by the inside of its L-shaped and use

certain parts of the tRNA, called identity elements, to distinguish these similar molecules

from one another.

Proofreading

Proofreading occurs when a synthetase carries out step 1 of the aminoacylation reaction

with the wrong, but chemically similar, amino acid. It will not carry out step 2, but will

hydrolyze the aminoacyl adenylate instead.

Aminoacyl-tRNA Synthetases catalyze linkage of the appropriate amino acid to each

tRNA. There are at least 20 different aminoacyl-tRNA synthetase.The reaction occurs in 2

steps.

In Step 1, an O atom of the amino acid a-carboxyl attacks the P atom of the initial

phosphate of ATP.

In Step 2, the 2' or 3' OH of the terminal adenosine of tRNA attacks the amino acid

carbonyl C atom.

Meccanismo Amminoacil-tRNA sintasi

L’enzima individua il specifico tRNA a cui legare l’amminoacido riconoscendo specifiche

zone nucleotidiche del tRNA.

21 enzimi AA-tRNA sintasi ognuno specifico per ogni amminoacido

Classes of Aminoacyl-tRNA Synthetases

-Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val (Generally the Larger Amino Acids)

-Class II: Ala, Asn, Asp, Gly, His , Lys, Phe, Ser, Pro, Thr (Generally the smaller amino

acids)

Main Differences between the two classes:

1.Structural differences. Class I are mostly monomeric, class II are dimeric.

2.Bind to different faces of the tRNA molecule 3. While class I acylate the 2’ hydroxyl of the

terminal Ado, class II synthetases acylate the 3’-OH

tRNA Recognition by Synthetases

There is normally a single aminoacyl tRNA synthetase for each amino acid, despite the

fact that there can be more than one tRNA, and more than one anticodon, for an amino

acid (genetic code).

Recognition of the appropriate tRNA by the synthetases is not mediated solely by the

anticodon, usually just a few bases are involved in recognition.

Canonical and non-canonical miRNA biogenesis pathways

In the canonical miRNA are transcribed by RNA pol II to produce pre-miRNA which is then

processed by the DROSHA complex to generate precursor miRNA.

Non canonical pathway use a spiceosome-dependent mechanism.

The diverse mechanisms of microRNA (miRNA) activity that reduce the mRNA activity.

miRNAs use two mechanisms to exert gene regulation.

miRNA involvement in cancer by modulation of expression of tumor suppressor genes and

oncogenes.

The sterol-regulatory element-binding p