EXPRESION GENICA EN LA CELULA ANIMAL

Note that a small proportion of nuclear RNA molecules can be converted naturally to cDNA by virally encoded and cellular reverse transcriptases, and thereafter integrate into chromosomal DNA at diverse locations. Note also that
the mitochondrion synthesizes its own rRNA and tRNA and a few proteins which are involved in the oxidative phosphorylation (Ox. phosph.) system. However, the proteins of mitochondrial ribosomes and the majority of the proteins in the mitochondrial oxidative phosphorylation system and other mitochondrial proteins are encoded by nuclear genes and translated on cytoplasmic ribosomes, before being imported into mitochondria

TRANSCRIPCION

Definicin Mecanismo por el cual el ADN transcribe su informacin a la molcula de ARN

Elementos Pentosa: Ribosa NTPs: U, A, C, G Enzima: ARNpol Molde: Molde ADN

TRANSCRIPCION
Caractersticas Direccin: 5 3 Antiparalela Complementaria Asimtrica

Tipos ARNm, ARNt, ARNr, ARNhn, ARNsn.

Reaccin Enlace Fosfodister 3 5. Primer nt conserva grupo trifosfato.

Estructura Espacial Primaria Secundaria Terciaria.

TRANSCRIPCION
Estructura Espacial Primaria Secundaria Terciaria.

TRANSCRIPCION Mecanismo por el cual el ADN se transcribe a ARN. Slo algunas regiones se transcribe. Una hebra de la hlice del ADN transcribe.
ARN
5

ARN
3

A
ARN

Reaccin Enlace fosfodister 35 entre los ribonucletidos: ATP, CTP, UTP, GTP.

Tipos ARNm ARNt ARNr ARNhn ARNsn

Ncleo, citoplasmtico, mitocondrial Ncleo Ncleo

PROMOTOR

INICIO

ELONGACION

FINALIZACIN

+1

PROCARIONTES Transcripcin y traduccin simultnea. Genes unidades continuas EUCARIONTES Transcripcin y traduccin separadas en espacio y tiempo. Genes fragmentados: intrones y exones
%Masa Celular Pared Bacteriana Membranas ADN ARNm ARNt ARNr # Molc./Cl Tipos Molc. # Copia por tipo molc.

10 10 1.5 1 3 16

1 2 1 1,500 200,000 38,000

1 2 1 600 60 2

1 1 1 2-3 >3,000 19,000

PROCESO DE TRANSCRIPCION

Various transcription factors (TF) are required to bind to a promoter sequence in the immediate vicinity of a gene , in order to subsequently position and guide the RNA polymerase that will transcribe the gene . Chain synthesis is initiated with a nucleoside triphosphate and chain elongation occurs by successive addition of nucleoside monophosphate residues provided by rNTPs to the 3 OH. This means that the 5 end will have a triphosphate group, which may subsequently undergo modification (e.g. by capping) and the 3 end will have a free hydroxyl group. Note that the sequence of the RNA will normally be identical to the sense strand of the gene (except U replaces T) and complementary to the template strand.

Cadena antisentido

5 3 5 -

-3 DNA - 5 - 3 mRNA

Cadena molde

CADENA SIMPLE (UNA HEBRA) CADENA COMPLEMENTARIA Y ASIMETRICA CADENA ANTIPARALELA

DIRECCION DE LA TRANSCRIPCIN: 5 3

TIPOS DE ARN POLIMERASA

EUCARIOTAS

Types
Nuclear I II

Inhibicin Amantina
Insensible Muy sensible

Genes transcribe
Localized in the nucleolus. A single primary transcript (45S rRNA) is cleaved to give the three rRNA classes listed Polymerase II transcripts are unique in being subject to capping and polyadenylation The promoter for some genes transcribed by RNA polymerase III (e.g. 5S rRNA, tRNA, 7SL RNA) is internal to the gene and for others (e.g. 7SK RNA) is located upstream

28S rRNA; 18S rRNA; 5.8S RNAr

ARNm; snRNA genes, ARN viral

III

Sensible

5S rRNA; tRNA genes; U6 snRNA; 7SL RNA; 7SK RNA; 7SM RNA

Mitocondrial PROCARIOTAS E. coli 2

Insensible

rARN, tARN, mARN

Sensible Rifampicina

Holoenzima (, , , 70, ) Centro cataltico Une al molde de manera inespecfica Se une a Sntesis especfica, Reconoce promotor. Varios tipos

PROCESO DE TRANSCRIPCION
Etapas 1. INICIO 2. ALARGAMIENTO 3. FINALIZACIN 4. MADURACION

INICIO (E. coli) Configuracin Cerrada: Unin al promotor del factor sigma Configuracin Abierta: Ruptura de los puentes de H; liberacin de ; Primer enlace fosfodiester ALARGAMIENTO Sntesis: 40nt/seg. ARNpol cubre 18 bases; en 12 hay formacin de duplex ADN-ARN FINALIZACION 1.Independiente de Rho: secuencia consenso palindrmica rica en GC precede a residuos de U. Estructura secundaria forma bucle. Protena Tau en fagos; NuvA en E.coli se une al sitio de de la ARNpol. 2. Dependiente de Rho, posee actividad ATPasica. Factor Rho actua sobre la ARNpol para liberarla.

PROCESO DE TRANSCRIPCION
Etapas 1. INICIO 2. ALARGAMIENTO 3. FINALIZACIN 4. MADURACION

INICIO (E. coli) Configuracin Cerrada: Unin al promotor del factor sigma Configuracin Abierta: Ruptura de los puentes de H; liberacin de ; Primer enlace fosfodiester ALARMIENTO Sntesis: 40nt/seg. ARNpol cubre 18 bases; en 12 hay formacin de duplex ADN-ARN FINALIZACION 1.Independiente de Rho: secuencia consenso palindrmica rica en GC precede a residuos de U. Estructura secundaria forma bucle. Protena Tau en fagos; NuvA en E.coli se une al sitio de de la ARNpol. 2. Dependiente de Rho, posee actividad ATPasica. Factor Rho actua sobre la ARNpol para liberarla.

TRANSCRIPCION EN PROCARIONTES La ARN polimerasa se une al factor de iniciacin , cambia de conformacin y se une a la zona promotora rica en A, T Liberacin del factor Desenrollamiento del ADN Lectura en sentido 3 5 y Sntesis de ARN en sentido 5 3 Terminacin de la sntesis al llegar a secuencia rica en G, C con la colaboracin del factor

El ARNm esta listo para la traduccin

Los ARNr y ARNt necesitan maduracin

TRANSCRIPCION EN EUCARIONTES

Hay tres ARN polimerasas especializadas en cada tipo de ARN. Cada ARNpol nuclear tiene varias unidades ARNpol y cloroplasto solo una unidad y sensible a la rifampicina

El ADN tiene que desbloquearse de las histonas antes de abrirse

Los factores de iniciacin estn a su vez controlados por activadores La transcripcin de los ARNr produce tpica configuracin de rbol en navidad. Se aade una caperuza de GTP metilado para indicar el extremo inicial del ARN y dar estabilidad a la molcula En el extremo final se aade una cola de poli A Todos los ARN sintetizados tienen proceso de maduracin

La maduracin utiliza ESPLICEOSOMAS RNPpn

Los ARNt tienen bases singulares y termina siempre con el triplete CCA

ESTRUCTURA DE LA REGION PROMOTORA

A G

Secuencias consenso A) Secuencia Pribnow (-10): Favorece la ruptura de enlace de Hidrogeno. Mutaciones afectan el rendimiento de sntesis de ARNm B) Secuencia TTGCACA (-35): Mutaciones inactivan a los Promotores

CIS-ACTING ELEMENTS RECOGNIZED BY UBIQUITOUS TRANSCRIPTION FACTORS

Cis element
GC box
TATA box

DNA sequence is identical to, or a variant of

Associated trans-acting factors

Comments

GGGCGG
TATAAA

Spl
TFIID

Spl factor is ubiquitous

TFIIA binds to the TFIIDTATA box complex to stabilize it Large family of trans-acting factors Genes activated in response to cAMP

CAAT box CRE (cAMP response element)

CCAAT GTGACGTA/CAA/G

Many, e.g. C/EBP, CTF/NFI CREB/ATF family, e.g. ATF-1

ESTRUCTURA DE LA REGION PROMOTORA

Alternative orientations for GC and CAAT box elements are indicated by chevron orientation: > = normal orientation; < = reverse orientation. The glucocorticoid receptor gene is unusual in possessing 13 upstream GC boxes (10 in the normal orientation; three in the reverse orientation). The tRNA genes are transcribed by RNA polymerase III and have an internal bipartite promoter comprising element A (usually within the nucleotides numbered +8 to +19 according to the standard tRNA nucleotide numbering system) and element B (usually between nucleotides +52 and +62). Specific transcription factors bind to these elements and then guide RNA polymerase III to start transcribing at +1.

CIS-ACTING SEQUENCES RECOGNIZED BY TISSUE-RESTRICTED AND TISSUE-SPECIFIC TRANSCRIPTION FACTORS

Consensus binding sequence (A/T)GATA(A/G) TGACTCAG GTTAATNATTAAC (= PE element) T(G/A)TTTG(C/T) GCCTGCAGGC (C/T)TAAAAATAA(C/T)3 (C/T)TA(A/T)AAATA(A/G) CAACTGAC ATGCAAAT (C/A)A(C/A)AG Transcription factor GATA-1, -2, etc. NF-E2 HNF-1 HNF-5 Ker1 MBF-1 MEF-2 MyoD OTF-2 TCF-1

Expression patterns
Erythroid cells Erythroid cells Differentiated liver, kidney, stomach, intestine, spleen Liver Keratinocytes Myocytes Myocytes Myoblasts + myotubes Lymphoid cells T cells

MADURACIN DE LOS ARN

1. ADICCION TERMINAL DE NUCLEOTIDOS Extremo 5: m7Gppp Extremo 3: poli(A) 2. MODIFICACION DE NUCLEOTIDOS METILACIN Extremo 5 Extremo 3 Exones 3. CLIVAJE SPLICING o Procesamiento

THE 5 END OF EUKARYOTIC mRNA MOLECULES IS PROTECTED BY A SPECIALIZED NUCLEOTIDE (CAPPING).

Primera Metilacin En el nitrgeno 7 de la Guanina invertida (5-5) En todos los ARNm. 7metil guanina transferasa CAP

Segunda Metilacin Frecuente en Eucariotas, ausente en eucariotas unicelulares. 2-oximetil transferasa.

2-O-metil transferasa
Tercera metilacin En 2-O.ribosa. Comn en 10-15% de los ARN con CAP
After the original gamma phosphate of the terminal 5 nucleotide is removed, a new GMP residue is provided by a GTP precursor which forms a specialized 5-5 triphosphate linkage with what was the terminal 5 nucleotide. Subsequent reactions lead to methylation of nitrogen atom 7 of the terminal G, and, in vertebrates, of the 2 carbon atom of the ribose of each of the two adjacent nucleotides. N, any nucleotide; Pu, purine.

El extremo 3 de la mayora de las ARNm de eucariotas esta poliadenilados

Seal de clivaje 3 upstream

15-30nt

Adicin ~200 Adeninas. Estabilidad del ARNm Evita degradacin con ayuda de PABP Remover poli(A) evita la traduccin in vivo De-adenilacin reduce la traduccin Los ARNm son almacenados sin poli(A) Facilitad aislamiento Enzima Poli(A) asociada a la PABP PABP: monmero de 70kDa, cubre 10-20 Adeninas Poli(A) polimerasa Protena PABP

The end of transcription of RNA polymerase II transcripts is signaled by a 3 cleavage in the transcribed RNA. In most mRNA species, this is achieved by an upstream AAUAAA signal in concert with, as yet, unidentified downstream signals. Cleavage occurs normally about 1530 nucleotides downstream of the AAUAAA element and AMP residues are subsequently added by poly(A) polymerase to form a poly(A) tail. Histone mRNA undergoes a different 3 cleavage reaction (see text).

PROCESAMIENTO DE

INTRONES

PROCESAMIENTO DE

INTRONES

RNA splicing involves endonucleolytic cleavage and removal of intronic RNA segments and splicing of exonic RNA segments

CONSENSUS SEQUENCES AT THE DNA LEVEL FOR THE SPLICE DONOR, SPLICE ACCEPTOR AND BRANCH SITES IN INTRONS OF COMPLEX EUKARYOTES

Highlighted nucleotides are almost invariant (but note that rare introns also exist where the conserved splice donor dinucleotide GT is replaced by AT and where the conserved splice acceptor dinucleotide AG is replaced by AC; see text). Other nucleotides represent the majority nucleotide found at this particular position. Note that in cases where pyrimidines (C/T or T/C) are preferred, no significance should be attached to which base comes first. For example, the consensus sequence of the branch site is written so as to highlight the similarity to the consensus branch site in yeast introns (TACTAAC), but the sequence given for the splice acceptor site does not signify a preference for C as opposed to T

MECHANISM OF RNA SPLICING (GU-AG INTRONS)

(A) Mechanism. The nucleophilic attack involves the 2-hydroxyl group attached to the conserved A at the branch site and the G of the conserved GU at the start of the intron, and results in a new covalent bond linking these two nucleotides to give a branched structure (lariat). (B) Role of snRNPs. Small nuclear ribonucleoprotein particles (snRNPs) are part of the spliceosome. U1 snRNA has a terminal sequence complementary to the splice donor consensus and binds to it by RNA-RNA base pairing. After the U1 snRNP has bound, U2 snRNA recognizes the branch site by a similar base-pairing reaction. Interaction between the splice donor and splice acceptor junctions is stabilized by subsequent binding of a preformed multi-snRNP particle, containing U4, U5 and U6 snRNAs, with the U5 snRNP able to bind simultaneously to both splice donor and splice acceptor

DROGAS QUE INHIBEN LA TRANSCRIPCION

ACTINOMICINA D Se une a residuos dGTP del ADN impidiendo el desplazamiento de la ARNpol en la etapa alargamiento
PROFLAVINA Se une al ADN molde RIFAMPICINA Inhibe a la ARNpol de procariotas y mitocondrial. Acta en la etapa de Inicio. Alfa-AMANTINA Inhibe a la ARNpol de eucariotas CORDICEPINA Inhibe la adiccin de poli(A).

BROMURO DE ETIDIO Se une al ADN.

-AMANTINA Inhibe a la ARNpol eucariotas

EXPRESSION OF THE HUMAN -GLOBIN GENE.

Exons 1 and 3 each contain noncoding sequences (shaded bars) at their extremities, which are transcribed and are present at the 5 and 3 ends of the globin mRNA, but are not translated to specify polypeptide synthesis. Such 5 and 3 untranslated regions (5 UTR and 3 UTR), however, are thought to be important in ensuring high efficiency of translation (see text). The stop codon UAA represents the first three nucleotides of the 3 untranslated region. Note that the initial translation product has 147 amino acids, but that the N-terminal methionine is removed by post-translational processing to generate the mature -globin polypeptide. The first two bases of the codon specifying Arg30 are encoded by exon 1 and the third base is encoded by exon 2 (i.e. intron 1 separates the second and third bases of the codon, an example of a phase 2 intron; see Box 14.3). The second intron separates codons 104 and 105 and is an example of a phase 0 intron; see Box 14.3, and also Figure 1.23 for an example of a phase 1 intron.

THE GENETIC CODE IS DECIPHERED BY CODON - ANTICODON RECOGNITION

The sequence of nucleotides in the mRNA sequence is interpreted from a translational start point (normally marked by the sequence AUG) and continues in the 5 3 direction until a stop codon is reached in that reading frame (see Figure 1.19). Each codon in the mRNA is recognized by the complementary anticodon sequence of a tRNA molecule to which a specific amino acid is covalently bonded to the adenosine at the 3 end (see insert).

ARN

TRANSFERENCIA

(ARNt)

Brazo Aceptor

CARACTERISTICAS
Tienen la similar forma y tamao (65-110 nt.) Represente el 10-15% del ARN total Coeficiente de Sedimentacin: 4S Peso Molecular: 22,000-37,300 Es el ARN con mayor # de bases modificadas Procesamiento en Eucariotas implica 10-19 bases En E. coli tiene 50 ARNts Enzima: aminoacilARNt sintetasa La aminoacilARNt sintetasa es especfica para reconocer el aa y el ARNt.
Brazo y Asa Anticodn Brazo y Asa D

Brazo y Asa TC

Asa Variable

PARTES
Extremo 3: termina en CCA. Se une al aa. Extrema 5: termina con Guanina Anticodon: Reconoce el ARNm Asa D: presenta Dehidroxiuridina. Reconoce a aminoacil sintetasa. Asa TCG: reconoce al ribosoma (ARN 5S). Tiene pseudouridina Asa Variable:

CARGADO DE AMINOACIDOS EN EL ARNt

TRANSCRIPCION EN EUCARIOTAS 1. 2. 3. 4. 5. Elementos Cis Elementos trans ADN policistronico Promotores mas distantes: 200-300pb. Secuencia Consenso (ARNpol II) TATA o ATA (-25, -30). Mas conservada CAAT o CAT (-70, -90). Menos conservada GC 6. Promotores Internos y factores de transcripcin (ARNpol III, sntesis de 5S t ARNt) 7. Intensificadores