celulas-e-estrutura-molecular-do-dna-aula-11.ppt ... · Fosfato P O O-O- O- ADP, CDP, GDP, TDP ATP,...

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Marcadores Biomoleculares Curso de Licenciatura em Biologia Curso de Licenciatura em Biologia Universidade Federal do Piau Universidade Federal do Piau Eleonidas Moura Lima pdfMachine - is a pdf writer that produces quality PDF files with ease! Get yours now! “Thank you very much! I can use Acrobat Distiller or the Acrobat PDFWriter but I consider your product a lot easier to use and much preferable to Adobe's" A.Sarras - USA

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Marcadores Biomoleculares

Curso de Licenciatura em BiologiaCurso de Licenciatura em Biologia

Universidade Federal do PiauUniversidade Federal do Piauíí

Eleonidas Moura Lima

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CCÉÉLULASLULAS

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ESTRUTURA DE NUCLEOTESTRUTURA DE NUCLEOTÍÍDEOS E DEOS E

ÁÁCIDOS NUCLCIDOS NUCLÉÉICOSICOS

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Fosfato

Purina ou pirimidina

H

Fosfato + açúcar (pentose) + base

Estrutura dos nucleotEstrutura dos nucleotíídeosdeos

Desoxirribonucleotídeo

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NucleosNucleosíídeodeo

pentose

Açúcar + base

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DesoxirriboseDesoxirribose

Pentose : 5 carbonos

1

23

4

5 Base

Fosfato

C que participa da ligação fosfodiéster

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Anéis heterocíclicos átomos de N dão caráter básico

1 único anel de 6 átomos 2 anéis fundidos de 5 e 6 átomos

Pirimidina Purina

As bases nitrogenadasAs bases nitrogenadas

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Timina (T) Adenina (A)

Citosina (C) Guanina (G)

As bases nitrogenadasAs bases nitrogenadas

Purinas : A, GPirimidinas : T, C

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FosfatoFosfato

P

O

O-O-

O-

ADP, CDP, GDP, TDP

ATP, CTP, GTP, TTP

AMP, CMP, GMP, TMP

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A letra "d" é utilizada para indicar que o açúcar é a desoxirribose.

-ácido uridílicouridinaUracil

dTMPácido timidílicotimidinaTimina

dCMPácido citidílicocitidinaCitosina

dGMPácido guanílicoguanosinaGuanina

dAMPácido adenílicoadenosinaAdenina

DNANucleotídioNucleosídioBase

Nomenclatura dos monômeros dos Nomenclatura dos monômeros dos áácidos nuclcidos nuclééicosicos

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LigaLigaçção ão fosfodifosfodiéésterster

OH ligado ao C 3�Nucleotídeo que será

incorporado a cadeia nascente

Extremidade 5� da cadeia polinucleotídica

Trifosfato ligado ao C 5�

2 fosfatos são

liberados

Sentido da síntese

5�

3�

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Os Os áácidos nuclcidos nuclééicosicosDNA

5�

3�

� Oligonucleotídeo : cadeia

pequena de nucleotídeos

(<50 nt)

� Polinucleotídeo : cadeia

acima de 50 nt

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O DNA armazena a informaO DNA armazena a informaçção genão genééticatica

Friederich Miescher (1863) : isolou uma substância contendo

fósforo � a �nucleína�

DNA (ácido) + proteína (básica)

A informação genética seria armazenada no DNA ou nas proteínas ?

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Estrutura do DNAEstrutura do DNA

Watson e Crick (1953) : propuseram o modelo da dupla hélice

Chargaff e cols. : lei da complementaridade

A=T e C=G A+C = T+Glogo

Franklin e Wilkins : DNA é helicoidal (hélice)

Padrões de difração de raios-X

Possui 2 periodicidades :

Primária : a cada 3,4 ÅSecundária : a cada 34 Å

3,4 Å

34 Å

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O modelo da dupla hO modelo da dupla héélice lice

2 cadeias de polinucleotídeos, unidas em espiral

Distância entre

nucleotídeos3,4 Å

36 Å Volta completa da hélice

10,5 bases (em meio aquoso)

Watson e Crick propuseram 10 bases por volta (34 Å)

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A Dupla HA Dupla Héélicelice

Fosfatos naporção externa -carga negativa

Cadeias antiparalelas

Ligação entre

nucleotídeos : ligação

fosfodiéster

Pareamento de bases é específico : A-T e C-GAs fitas são complementares

Ligação entre

as bases :

pontes H

5�

5�3�

3�

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Formas tridimensionais do DNAFormas tridimensionais do DNA

Forma A Forma B Forma Z

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Propriedades quPropriedades quíímicas dos micas dos áácidos nuclcidos nuclééicosicos

DNA dupla hélice

Denaturação Reassociação (annealing)

DNA parcialmente denaturado

Separação das

fitas

Fitas de DNA separadas estruturam-se casualmente em espirais

Associação das fitas por

pareamento de bases

↑ To ou ▲pH (< 3,0 ou > 10,0)

Ruptura das pontes de H

Denaturação e renaturação

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Temperatura desnaturação

Metade das moléculas de DNA estão

desnaturadas

Varia de acordo com o tamanho e a

composição das moléculas :

Temperatura de melting (TM)

Quanto maior a molécula Quanto maior o conteúdo G-C

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HibridaHibridaçção :ão : ácidos nucléicos de origem diferente podem se associar

Amostra 1 Amostra 2

Dúplex híbrido

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Transferência da informaTransferência da informaçção genão genééticatica

DNA RNA PROTEÍNA

transcrição tradução

duplicação

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DUPLICAÇÃO DO DNA

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Watson e Watson e CrickCrick (1953)(1953)

Modelo da Dupla Hélice

Sugeria duplicação semi-

conservativa

Fitas velhas - molde

Fitas novas

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Hipótese 1

Semi-conservativa

Hipótese 2 Hipótese 3

Conservativa Dispersiva

Modelos de duplicaModelos de duplicaçção do DNAão do DNA

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MeselsonMeselson e e StahlStahl (1958)(1958)

meio com N pesado (15N)

meio com N leve (14N)

DNA pesado (15N)

DNA híbrido (15N/ 14N)

DNA leve (14N)DNA híbrido

Molécula mãe original

Primeira geração de moléculas

filhas

Segunda geração de moléculas

filhas

Centrifugação em

gradiente de densidade de CsCl

Replicação conservativa

1a geração

Replicação dispersiva

1a geração 2a geração

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DuplicaDuplicaçção em ão em procariotosprocariotos

Para as fitas velhas funcionarem como molde, a dupla hélice precisa ser aberta

Várias origens ou uma única ?

Cairns, 1963

Em procariotos, existe uma única origem de duplicação

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Sentido da duplicaSentido da duplicaççãoão

Bidirecional

Unidirecional

Cairns, 1963

Pulsos de timidina H3 antes do término da duplicação :

marcação radioativa

visualizada nas duas forquilhas de duplicação

Forquilha de

duplicação

Forquilha de

duplicação

A duplicação é bidirecional

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Seqüências de 13 pbSítios de ligação da proteína DnaA

Seqüências de 9 pb

Ricas em AT

Origem da replicação : Seqüência específica para ligação de

proteínas iniciadoras (promovem abertura da dupla hélice)

Menor gasto de energia para separar as fitas

A A sequênciasequência da origem da origem éé casual ou existe uma casual ou existe uma sequênciasequência consenso ?consenso ?

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Uma vez que a dupla hUma vez que a dupla héélice estlice estáá aberta, aberta, como como éé feita a sfeita a sííntese da nova fita ?ntese da nova fita ?

DNA DNA polimerasespolimerases

Kornberg (1957) : DNA polimerase I

Requerimentos da DNA pol I para sintetizar nova fita de DNA :

Deoxinucleotídeos trifosfatados

Íons Mg2+

DNA pré-existente, para servir como molde

Uma extremidade OH-3� de uma cadeia de DNA pré-existente

para realizar a ligação fosfodiéster

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Extremidade OH-3� livre da nova fita de DNA

Extremidade 5�da nova fita de

DNA

Extremidade 5�da nova fita de

DNA

DNA pol I

LigaLigaçção ão fosfodifosfodiéésterster

A síntese ocorre sempre no sentido 5� 3�

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Primer

O problema da iniciaO problema da iniciaççãoão

DNA pol I não consegue iniciar a síntese da nova fita � ausência

de extremidade OH-3� livre

Primase : sintetiza oligonucleotídeo (primer) de RNA, fornecendo a extremidade OH-3� livre para a DNA pol I

5� 3�3� 5�

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A DNA pol I possui outras atividades enzimA DNA pol I possui outras atividades enzimááticas :ticas :

Atividade exonucleásica 5�-3�

Atividade exonucleásica 3�-5�

Nuclease : degrada ác. nucleicoExonuclease : cliva extremidadesEndonuclease : cliva ligações internas

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Outras DNA Outras DNA polimerasespolimerases foram descobertas :foram descobertas :

DNA pol II :

� Reparo do DNA

� Atividade polimerase 5�-3�

� Atividade exonuclease 3� � 5�

DNA pol III :

� Possui várias subunidades

� Atividade polimerase 5�- 3�

� Atividade exonuclease 3� � 5�

DNA polimerase IV e V � envolvidas em diferentes processos de reparo

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Processividade : habilidade da enzima de repetir a sua

função catalítica sem dissociar-se do seu substrato

Definida pelo no médio de nt incorporados sem que a DNA

pol se dissocie do DNA molde

Para a DNA pol :

As DNA pol diferem em processividade

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DNA pol III : a enzima responsDNA pol III : a enzima responsáável pela vel pela replicareplicaçção em ão em procariotosprocariotos

A holoenzima DNA pol III

Núcleo catalítico mínimo

Liga dois núcleos

catalíticos

Mantém a enzima ligada ao DNA

: polimerase : proofreading

Complexo �clamp-loading�

Subunidade : aumenta a processividade da DNA pol III

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DuplicaDuplicaççãoão do DNA : um do DNA : um processoprocesso de de altaaltafidelidadefidelidade

E. coli � 1 erro a ~ cada 109 bases adicionadas ou a após mil ou 10 mil eventos de replicação

DNA polimerase tem um sítio ativo que só se fixa

quando as bases estão pareadas corretamente

DNA polimerase tem capacidade de verificar a fidelidade

da replicação na cadeia nascente (proofreading) e corrigir

os erros (atividades exonucleásicas)

Depois da replicação, existe um sistema de reparo que

corrige qualquer erro de pareamento de bases

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DuplicaDuplicaççãoão do DNA : um do DNA : um processoprocesso de de altaaltafidelidadefidelidade

Pareamento incorreto :

� Geometria

diferente

� Não acomoda-

se no sítio ativo

da DNA pol

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DNA pol I

Sítio ativo da atividade exonucleásica3�5� (proofreading)

Sítio ativo da atividade polimerase

Forma rara da CPareia-se com A

C na forma rara volta para a forma normal � pareamento inadequado

Bloqueio da duplicação

Pareamento errôneo localizado no sítio da atividade exonucleásica

O nucleotídeo mal pareado é removido

DNA pol reassume a atividade polimerase

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Direção em que as fitas

se estendem

3�

3�5�

5�

5�

3�5�

3�DNA pol só age no sentido 5�- 3� !!!

Direção em que as fitas

se estendem

3�

3�5�

5�

5�

3�5�

3�

A síntese é contínua numa fita e descontínua na outra...

Fita líder (contínua)

Fita descontínua

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SSííntese dos ntese dos primersprimers na fita descontna fita descontíínuanua

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Abertura e desenrolamento do DNAAbertura e desenrolamento do DNA

TopoisomeraseHelicase

SSBs (single strandbinding proteins)

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InIníício da duplicacio da duplicaççãoãoOri C

13 pb 9 pb

Dna A une-se às repetições de 9 pb

Formação de um

complexo de Dna A

Fitas se separam na região

das repetições de 13 pb

Dna C Dna B (helicase)

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3�3�

3�5�

5�

TopoisomeraseHelicase

SSBs

5�

Holoenzima DNA pol III

Primase

DNA pol I

Ligase

InIníício e cio e elongamentoelongamento

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DuplicaDuplicaçção em ão em eucariotoseucariotos

O ciclo celular

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Genoma humano � ~ 10 mil locais de origem (maior velocidade de replicação)

VVáárias origens de duplicarias origens de duplicaççãoão

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Tipos de DNA polimerases (Eucariotos)

�� POLIMERASE POLIMERASE áá

�� POLIMERASE POLIMERASE ää

�� POLIMERASE POLIMERASE åå

�� RPARPA

�� POLIMERASE POLIMERASE ãã

-- NNúúcleocleo-- AtividadeAtividade de de polimerizapolimerizaççãoão e e primaseprimase-- SemSem atividadeatividade exonucleaseexonuclease 33��--55��

FitaFita descontdescontíínuanua

-- NNúúcleocleo-- AtividadeAtividade de de polimerizapolimerizaççãoão e e exoexo 33��--55��-- SemSem atividadeatividade primaseprimase

-- NNúúcleocleo-- AtividadeAtividade de de exonucleaseexonuclease 33��--55�� (~ DNA (~ DNA PoliPoli I)I)-- ReparoReparo de de lesõeslesões provocadasprovocadas porpor UVUV

-- NNúúcleocleo-- AtividadeAtividade semelhantesemelhante àà dasdas SSB de SSB de procariotosprocariotos

-- MitocôndriaMitocôndria

-- ReplicaReplicaççãoão do do genomagenoma mitocondrialmitocondrial

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O aparato de replicaO aparato de replicaçção ão eucariotoeucarioto

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5�3�

5�

3�

3�

5�

3�5�

DNA pol.

3�gap

O problema da duplicaO problema da duplicaçção no final da fita do DNAão no final da fita do DNA

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A A telomerasetelomerase

TelomeraseTemplate interno

Polimerização

Translocação

Nova polimerização

Fita molde

Fita nova

primase

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TranscriTranscriççãoão

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Bases

DNA : A, C, G e T

RNA : A, C, G e U

TranscriTranscriççãoão

Síntese dos diferentes tipos de RNA a partir de um molde de DNA, de acordo com

as regras da complementaridade

G≡C

A=U

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O princO princíípio da transcripio da transcriçção ão

Gene de interesse

DNA dupla fita As fitas são separadas

num local determinado

A fita de RNA é sintetizada

Fita molde do DNA

Fita simples de RNA

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� RNAt � leva aminoácidos para o ribossomo

� RNAr � principal componente dos ribossomos

� RNAm - taxa de renovação rápida � molde para síntese protéica

� snRNA � componentes do spliceossomo, responsável pelo

processamento do RNA

� iRNA � Envolvido na regulação da expressão gênica

Tipos de molTipos de molééculas de RNAculas de RNA

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TRANSCRIÇÃOREPLICAÇÃO

1 cromossomo inteiro écopiado

Requer primer

As duas fitas do DNA servem como molde

Somente 1 gene ou um grupo de genes

(regiões específicas do DNA) são

copiados

Não requer primer

Somente uma das fitas serve como

molde

Algumas regiões do DNA genômico

nunca são transcritas

Seqüências específicas regulam a

expressão

X

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� Catalisa reações fosfodiéster

� Tem atividade dependente da presença de DNA;

� A ordem em que os ribonucleotídios serão adicionados depende da

sequência de bases no DNA obedecendo a combinação: A=U e G=C

� Os precursores são os ribonucleosídeos trifosfatados (ATP, GTP, CTP, UTP) que utilizam a energia armazenada na ligação pirofosfato de cada nucleotídio ingressante para estabelecer a ligação fosfodiéster entre os monômeros do RNA em síntese

� Esta reação necessita da presença dos ions Mg2+ e Zn2+

RNA polimerase

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Abertura para Transcrição~ 17 pb

Fita não-codificadora(antisense)

Desenrola

Enrola

Fita codificadora (sense)

Sentido da transcriçãoSíntese no sentido 5'-3',

(~ alongamento de uma

cadeia de DNA)

TranscriTranscriçção : visão geralão : visão geral

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Central enzimática - 5 subunidades(2á, ,� e ù)

Direciona a enzima para região promotora

Não participa da fase de extensão

transiente e variável

Proteína multimérica constituída de cadeias polipeptídicas diferentes

Ligações entre estas moléculas são fracas

Quando todas estão associadas RNA polimerase holoenzima

TranscriTranscriçção em ão em procariotosprocariotos

RNA polimerase

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Up-stream Down-stream

UnidadeUnidade de de TranscriTranscriççãoão

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Estrutura da região promotora em E. Estrutura da região promotora em E. colicoli

Vários promotores em E. coli

Sequências consenso

-35 : TTGACA (reconhecida pela subunidade )

-10 : TATAAT

1a base (+1) : geralmente uma purina (A, G)

� Estão separadas entre si por cerca de 15-20 nucleotídios

� Posição dá orientação ao promotor direciona RNA pol

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IniciaIniciaçção da transcrião da transcriççãoão

Holoenzima RNA pol

Promotor Gene

A holoenzima RNA pol une-se não-especificamente ao DNA

A holoenzima procura pelo promotor

Quando o promotor é encontrado, a holoenzima e o promotor formam um complexo fechado

Uma alteração conformacionalproduz uma bolha de iniciação. Um

pequeno RNA é sintetizado

A subunidade se dissocia do núcleo enzimático e a RNA pol sai do promotor

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Extensão do transcritoExtensão do transcrito

Extremidade 5´fica disponível para a tradução � ocorre simultaneamente em procariotos

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TTéérmino da transcrirmino da transcriççãoão

Características do terminal Rho-independente

Pareamento com A é fracoFacilita liberação da fita quando a RNA pol parar

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CaracterCaracteríísticas do terminal sticas do terminal RhoRho--dependentedependente

� Região de simetria dupla no terminal com baixa complementaridade

entre as bases grampo "fraco" no RNA recém sintetizado

� Não existe uma seqüência rica em A após a dupla simetria

� Término da transcrição precisa de uma proteína auxiliar Rho (ñ)

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TranscriTranscriçção em ão em EucariotosEucariotos

Eucariotos x Procariotos

Núcleo CitoplasmaMonogênico Poligênico

Três RNA pol Uma RNA pol

RNA é modificado RNA não é mod.Tradução não é simultânea Tradução simultânea

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RNA RNA polimerasespolimerases em em eucariotoseucariotos

RNA polimerase I (RNAP I) - rRNA

RNA polimerase II (RNAP II) - mRNAs e snRNAs

RNA polimerase III (RNAP III) - tRNAs, 5S rRNA e outros RNAs pequenos

Fatores gerais de transcrição

Várias subunidades (10-15) com funções específicas na transcrição

Fatores de transcrição (TF) não fazem parte da holoenzima.

TF ligam-se previamente ao DNA e depois as RNA polimerases

TF são específicos para cada RNAP (TFI, TFII e TFIII)

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Regiões promotoras em Regiões promotoras em eucariotoseucariotos

Oct

Sequências consenso (RNA pol II)

- CAAT box : GGCCAATCT

- TATA box : TATAAAA

- Octâmero : ATTGCAT

GC box : GGGCGG

eElementos A e B

(RNA pol III)

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B

FormaFormaçção do aparato basal de transcrião do aparato basal de transcriççãoão

+1

Início da transcrição

TATA

+1TATATFIID

+1TATATFIIDARNA pol IIF

B +1TATATFIIDARNA pol IIF

E

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ElongamentoElongamento e Terminae Terminaççãoão

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Processamento do RNAmProcessamento do RNAm

Sítio Poli (A)

Sítio de terminação

Transcrição

RNA splicing

Clivagem por endonuclease

Poliadenilação

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SplicingSplicing

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�Proteção do mRNA

� Ligação do mRNA ao ribossomo

FormaFormaçção do cap 5ão do cap 5��

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AdiAdiçção da cauda poli Aão da cauda poli A

1) Facilitar a exportação

2) Manter a estabilidade do mRNA no citoplasma

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TranscriTranscriçção e processamento do ão e processamento do RNArRNAr

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Processamento do Processamento do RNAtRNAt

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