Elementos da transcrição celular

Prévia do material em texto

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O processo de Transcrição 
O Genoma em expressão 
Tópicos abordados 
•  A hipótese do RNA mensageiro 
•  Estrutura da unidade transcricional 
•  O transcrito e sua abundância 
•  A RNA polimerase 
 
 
 
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Manuscritos de Crick 
Crick, F.H.C. (1956): On Protein 
Synthesis. Symp. Soc. Exp. Biol. XII, 
139-163. 
Para Francis Crick, tanto RNA quanto proteína eram produzidos a 
partir da informação genética do DNA 
RNA como intermediário 
•  François Jacob e Matthew Meselson trabalhando juntos 
descobrem que as proteínas são produzidas nos ribossomos 
•  Mas era sabido que os genes estão nos cromossomos 
•  Portanto deve haver uma conexão, um jeito de transferir 
informação, entre o DNA e os ribossomos 
•  Surge ai a hipótese do RNA mensageiro 
Ideas on Protein Ssnthesis (Oct. 1956) 
The Doctrine of the Triad. 
The Central Dogma: "Once information has got into a protein it 
can't get out again". Information here means the sequence of 
a the amino acid residues, or other sequences related to it. 
That fs, we may be able to have 
Protein 
but never 
DNA '/-"--- L 
/---- ---i~Protein 
where the arrows show the transfer of information. 
Requirements for protein synthesis. 
( > a a passive template i.e. one which does not turn over 
in the proeess. This can be RNA 2 DNA. 
04 mixed intermediates of ribose nucleotides and amino IS ,' 
acids. The most favoured ones have the general formula: 
Base base base 
Sugar - phos - sukar - phos - sukar - phos - phos - amino acid 
DNA makes DNA by a special process not involving RNA and only 
involving proteins is a non-template manner (e.g. as enzymes, or 
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Hipótese do RNA mensageiro 
•  Um gene, um ribossoma, uma proteína? 
•  Sydney Brenner, Francois Jacob, e Matthew Meselson 
-> Hipótese do mRNA 
X 
A procura da enzima 
•  Identificação de uma RNA polimerase 1959 
–  Samuel Weiss e Leonard Gladstone 
–  Michael Chamberlin e Jerard Hurwitz 
•  1993 estrutura da RNA pol procariótica 
•  1999 estrutura da RNA pol de levedura 
•  2006 Premio Nobel para Roger Kornberg 
Cell 127, December 15, 2006 ©2006 Elsevier Inc. 1089
target of structural study 
was indeed the active form 
of RNA polymerase II. First, 
Kornberg and coworkers 
realized that TFIIH, a ten-
subunit helicase/kinase 
required to melt promoter 
DNA and phosphorylate 
RNA polymerase II, sepa-
rated into subcomplexes 
during purification and 
required special treatment 
to minimize dissociation. 
Second, the group discov-
ered that activators needed 
another multisubunit com-
plex they called Mediator to 
promote transcription ini-
tiation. Genetic dissection 
of the yeast transcription 
machinery by many labs 
was invaluable to Korn-
berg’s research, but this 
proved especially true for Mediator. 
Rick Young’s work at MIT identifying 
suppressors of partial deletions of 
the C-terminal repeat domain on RNA 
polymerase II yielded a set of pro-
teins termed SRBs, initially thought 
to be components of TFIID. By the 
mid 1990s, however, it became clear 
that Kornberg’s Mediator contained 
the SRBs. Although initially Mediator 
was considered a peculiarity of yeast, 
its mammalian homologs are now 
well established. Finally, in 1990, Al 
Edwards in the Kornberg lab realized 
that the purified yeast RNA polymer-
ase II contained substochiometric lev-
els of subunits RPB4/7. Using a yeast 
strain lacking RPB4 and an anti-RNA 
polymerase II monoclonal antibody 
developed by Dick Burgess in Wis-
consin, Edwards was able to obtain 
yeast RNA polymerase II pure and 
homogeneous enough to form large, 
well-ordered crystals. Darst and 
Edwards then used the new positively 
charged lipid method to produce 2D 
crystals of yeast RNA polymerase II 
and a 16 Å electron crystal structure 
in 1991 (Darst et al., 1991).
At this point, the group realized that 
the 2D crystals might yield 3D crystals 
if used as seeds in crystallization tri-
als. Immediately, Edwards set up the 
experiment, yielding 3D crystals in 
short order, and, by 1993, the first dif-
fraction patterns were in hand. Then 
came the tedious effort of produc-
ing isomorphous crystals, improving 
the diffraction, and obtaining heavy-
metal derivatives for phasing of the 
diffraction pattern. Again, success 
relied both on making connections 
to advances by other researchers 
and on the group’s persistence and 
experimental rigor. The construction 
of high-intensity X-ray sources from 
synchrotron beamlines (especially 
at the Stanford Linear Accelerator, 
where the group had ready access) as 
well as the development of improved 
computational methods for crystal-
lography, made the work possible. 
The size of RNA polymerase II meant 
that conventional heavy metals were 
too small for phasing; success came 
from systematic screening of various 
heavy metal clusters (e. g., W18) to 
find those that would bind appropri-
ately. Jianhua Fu and David Bushnell 
were the unsung heroes in this story 
for their persistence in finding deriva-
tives that allowed solution of the 
structure (Fu et al., 1999).
Just as Kornberg’s work on RNA 
polymerase II was coming to frui-
tion, Seth Darst, who had moved to 
Rockefeller in 1993, obtained a 3.4 Å 
resolution crystal structure of bacte-
rial RNA polymerase from Thermus 
aquaticus (Zhang et al., 1999). Darst’s 
breakthrough work gave 
the first high-resolution 
view of RNA polymerase, 
revealing the remarkable 
complexity with which the 
two large subunits formed 
a nucleic-acid binding cleft. 
Simultaneous publication 
of a 5 Å resolution structure 
of yeast RNA polymerase II 
(Fu et al., 1999) showed the 
remarkable similarity of the 
two enzymes. Joined by 
Averell Gnatt and later by 
Patrick Cramer, Kornberg’s 
team finished the high-res-
olution structure, obtaining 
not only a 2.8 Å resolution 
structure of the ten-subunit 
yeast RNA polymerase II 
lacking RPB4/7 (Figure 2) 
but also a 3.4 Å resolution 
structure of its transcrib-
ing complex showing the locations of 
RNA and DNA in the active site and 
the nine-base pair RNA-DNA hybrid 
in the main channel (Cramer et al., 
2001; Gnatt et al., 2001).
Lessons from the RNA Polymer-
ase Crystal Structure
Entry into the 21st century proved to 
be a watershed in the hunt for the 
structural basis of transcription. New 
insights into the molecular basis of 
transcription using structural analysis 
of RNA polymerase and its complexes 
prompted other research groups to 
join the effort, and many new struc-
tures appeared (Davis et al., 2002; 
Murakami et al., 2002; Vassylyev et 
al., 2002; Kettenberger et al., 2003; 
Bushnell et al., 2004; Westover et al., 
2004; Tuske et al., 2005). Space and 
citation limits preclude a complete 
accounting, but these structures 
included views of RNA polymerase at 
increased resolution, with bound NTP 
substrates, in complex with inhibitors 
and in complex with basal initiation 
factors, including  factor from bac-
teria and Mediator, TFIIB, TFIIS, and 
TFIIF from yeast. Among these, sev-
eral key structures have been pro-
duced by the Kornberg group and its 
alumni, including a key snapshot of 
the catalytic center poised for cataly-
sis, which appeared in the December 
Figure 2. The 2.8 Å Resolution Crystal Structure of Yeast 
RNA Polymerase II Lacking Subunits RPB4 and RPB7
The bridge helix extends across the main cleft in this view, which 
shows the upstream face of RNA polymerase II. The mobile clamp 
domain is formed by the segments of RPB1 in the lower left and a 
small portion of RPB2. Some of the loops that extend into the cleft or 
the channels of the enzyme are visible. A Mg2+ ion (magenta sphere) 
marks the position of the catalytic center. Reprinted with permissionfrom Cramer et al., 2001, copyright 2006 AAAS.
Leading Edge
Essay
Cell 127, December 15, 2006 ©2006 Elsevier Inc. 1087
Roger Kornberg (Figure 1) has spent 
three decades studying how DNA 
is packaged in eukaryotic cells and 
how RNA polymerase II, the central 
enzyme of transcription, extracts 
genetic information from this DNA. He 
achieved the central breakthrough in 
2001 with crystal structures of yeast 
RNA polymerase II alone and in a 
transcribing complex with its DNA 
template and RNA product (Cramer 
et al., 2001; Gnatt et al., 2001). Many 
factors lay behind this breakthrough, 
including Kornberg’s keen insight, 
the hard work of a talented group 
of postdocs and graduate students, 
and advances in crystallographic 
tools that brought large biomolecules 
within reach. Key among these was 
the choice to work with a eukaryotic 
microbe, the budding yeast Saccha-
romyces cerevisiae, which is amena-
ble to powerful genetics and large-
scale biochemistry. Kornberg’s path 
to the Nobel-winning discoveries 
beautifully illustrates how the most 
important fruits of scientific discov-
ery are almost never the low-hanging 
ones, but instead grow from years of 
rigorous experimental practice and 
from pulling together multiple diverse 
threads of research.
RNA Polymerase: The Engine of 
Gene Expression
As the first step in the expression 
of genetic information, transcription 
is among the most highly regulated 
biological processes. Transcriptional 
regulation is a primary determinant 
of homeostatic, adaptive, and devel-
opmental cellular functions when it 
operates normally and of disease 
states when it goes awry or is co-
opted by pathogens.
Since formulation of the central 
dogma of molecular biology half a cen-
tury ago, a full understanding of the 
molecular machinery of transcription 
and its regulation has been a prized 
goal. The key enzyme in this process, 
RNA polymerase, was discovered in 
1959 by Samuel Weiss and Leonard 
Gladstone in extracts of mammalian 
cells (Weiss and Gladstone, 1959). 
Shortly thereafter, RNA polymerase 
was purified from different species 
of bacteria by Michael Chamberlin, 
Jerard Hurwitz, and others. It took 
another decade for researchers, most 
notably Robert Roeder, to tease out 
the existence of three separate forms 
of RNA polymerase in eukaryotes. 
These different forms divide respon-
sibilities for synthesis of rRNA (RNA 
polymerase I), mRNA (RNA polymer-
ase II), and tRNA (RNA polymerase 
III) (Roeder and Rutter, 1969).
Studies in the final three decades 
of the twentieth century amassed a 
wealth of information about the subu-
nit composition, biochemical proper-
ties, and accessory factors of RNA 
polymerase. Several key insights 
emerged that proved remarkably 
concordant with the crystal struc-
tures of bacterial RNA polymerase, 
yeast RNA polymerase II, and its tran-
scribing complexes, when the struc-
tures became available. First, RNA 
polymerases from bacteria to humans 
contain an essential and highly con-
served core of two large subunits 
(called  and  in bacteria and RPB1 
and RPB2 in eukaryotic RNA polymer-
ase II), and smaller subunits ( dimer 
and  in bacteria; RPB3/11 and RPB 
6 in RNA polymerase II) that stabilize 
association of the two large subunits. 
Additional small subunits of varying 
essentiality associate with the core 
enzyme (RPB4, 5, 7, 8, 9, 10, and 12 
in RNA polymerase II). The two large 
subunits form the catalytic center 
and make all essential contacts to the 
nucleic-acid scaffold. This scaffold 
consists of a ?9 base pair RNA:DNA 
hybrid within a ?14 base pair melted 
DNA bubble, ?7 nucleotides of sin-
A Long Time in the Making— 
The Nobel Prize for RNA Polymerase
Robert Landick1,*
1Department of Bacteriology, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706, USA
*Contact: landick@bact.wisc.edu
DOI 10.1016/j.cell.2006.036
The 2006 Nobel Prize in Chemistry has been awarded to Roger Kornberg for elucidating the 
molecular basis of eukaryotic transcription. The prize caps a decades-long quest to unlock 
one of the central mysteries of molecular biology—how RNA transcripts are assembled.
Figure 1. Twenty years of diligence pays off
Roger Kornberg has been awarded this year’s 
Nobel Prize in Chemistry for his decades-long 
dedication to elucidating the molecular un-
derpinnings of eukaryotic transcription and 
the part played by RNA polymerase II. Photo 
courtesy of L.A. Cicero/Stanford University 
News Service. 
Leading Edge
Essay
Cell 127, December 15, 2006 ©2006 Elsevier Inc. 1087
Roger Kornberg (Figure 1) has spent 
three decades studying how DNA 
is packaged in eukaryotic cells and 
how RNA polymerase II, the central 
enzyme of transcription, extracts 
genetic information from this DNA. He 
achieved the central breakthrough in 
2001 with crystal structures of yeast 
RNA polymerase II alone and in a 
transcribing complex with its DNA 
template and RNA product (Cramer 
et al., 2001; Gnatt et al., 2001). Many 
factors lay behind this breakthrough, 
including Kornberg’s keen insight, 
the hard work of a talented group 
of postdocs and graduate students, 
and advances in crystallographic 
tools that brought large biomolecules 
within reach. Key among these was 
the choice to work with a eukaryotic 
microbe, the budding yeast Saccha-
romyces cerevisiae, which is amena-
ble to powerful genetics and large-
scale biochemistry. Kornberg’s path 
to the Nobel-winning discoveries 
beautifully illustrates how the most 
important fruits of scientific discov-
ery are almost never the low-hanging 
ones, but instead grow from years of 
rigorous experimental practice and 
from pulling together multiple diverse 
threads of research.
RNA Polymerase: The Engine of 
Gene Expression
As the first step in the expression 
of genetic information, transcription 
is among the most highly regulated 
biological processes. Transcriptional 
regulation is a primary determinant 
of homeostatic, adaptive, and devel-
opmental cellular functions when it 
operates normally and of disease 
states when it goes awry or is co-
opted by pathogens.
Since formulation of the central 
dogma of molecular biology half a cen-
tury ago, a full understanding of the 
molecular machinery of transcription 
and its regulation has been a prized 
goal. The key enzyme in this process, 
RNA polymerase, was discovered in 
1959 by Samuel Weiss and Leonard 
Gladstone in extracts of mammalian 
cells (Weiss and Gladstone, 1959). 
Shortly thereafter, RNA polymerase 
was purified from different species 
of bacteria by Michael Chamberlin, 
Jerard Hurwitz, and others. It took 
another decade for researchers, most 
notably Robert Roeder, to tease out 
the existence of three separate forms 
of RNA polymerase in eukaryotes. 
These different forms divide respon-
sibilities for synthesis of rRNA (RNA 
polymerase I), mRNA (RNA polymer-
ase II), and tRNA (RNA polymerase 
III) (Roeder and Rutter, 1969).
Studies in the final three decades 
of the twentieth century amassed a 
wealth of information about the subu-
nit composition, biochemical proper-
ties, and accessory factors of RNA 
polymerase. Several key insights 
emerged that proved remarkably 
concordant with the crystal struc-
tures of bacterial RNA polymerase, 
yeast RNA polymerase II, and its tran-
scribing complexes, when the struc-
tures became available. First, RNA 
polymerases from bacteria to humans 
contain an essential and highly con-
served core of two large subunits 
(called  and  in bacteria and RPB1 
and RPB2 in eukaryotic RNA polymer-
ase II), and smaller subunits ( dimer 
and  in bacteria; RPB3/11 and RPB 
6 in RNA polymerase II) that stabilize 
association of the two large subunits. 
Additional small subunits of varying 
essentiality associatewith the core 
enzyme (RPB4, 5, 7, 8, 9, 10, and 12 
in RNA polymerase II). The two large 
subunits form the catalytic center 
and make all essential contacts to the 
nucleic-acid scaffold. This scaffold 
consists of a ?9 base pair RNA:DNA 
hybrid within a ?14 base pair melted 
DNA bubble, ?7 nucleotides of sin-
A Long Time in the Making— 
The Nobel Prize for RNA Polymerase
Robert Landick1,*
1Department of Bacteriology, 420 Henry Mall, University of Wisconsin-Madison, Madison, WI 53706, USA
*Contact: landick@bact.wisc.edu
DOI 10.1016/j.cell.2006.036
The 2006 Nobel Prize in Chemistry has been awarded to Roger Kornberg for elucidating the 
molecular basis of eukaryotic transcription. The prize caps a decades-long quest to unlock 
one of the central mysteries of molecular biology—how RNA transcripts are assembled.
Figure 1. Twenty years of diligence pays off
Roger Kornberg has been awarded this year’s 
Nobel Prize in Chemistry for his decades-long 
dedication to elucidating the molecular un-
derpinnings of eukaryotic transcription and 
the part played by RNA polymerase II. Photo 
courtesy of L.A. Cicero/Stanford University 
News Service. 
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4 
Conteúdo de RNA na célula 
K. pastoris 
rRNA maior (26S; 2,6 kb) 
rRNA menor (16S; 1,5 kb) 
rRNA 5S; 127 nucleotídeos 
Glicerol Glicose 
rRNA maior 
rRNA menor 
RNA total extraído de células de 
Pichia pastoris e analisado por 
eletroforese em gel de agarose 
corado com brometo de etídeo 
O RNA ribossômico é de longe 
o mais abundante na célula ! 
Tipo de RNA abundância 
rRNA 80% 
tRNA 15% 
mRNA 5% 
Outros RNAs <1% 
S. Cereviseae em crescimento rápido. 
Modificado de Warner Trends in 
Biochemical Science 24, 437 (1999) 
RNA e o Dogma Central 
Proteínas 
regulatório 
estrutural 
enzimático 
codificador 
Não codificador 
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Universo de RNA 
Table 6-1 Molecular Biology of the Cell (© Garland Science 2008) 
Por que Dogma Central? 
“As it turned out, the use of the word dogma caused almost more trouble than 
it was worth. Many years later Jacques Monod pointed out to me that I did not 
appear to understand the correct use of the word dogma, which is a belief that 
cannot be doubted. I did apprehend this in a vague sort of way but since I 
thought that all religious beliefs were without foundation, I used the word the 
way I myself thought about it, not as most of the world does, and simply 
applied it to a grand hypothesis that, however plausible, had little direct 
experimental support." 
Crick, Francis (1988) in What Mad Pursuit: A Personal View of Scientific Discovery 
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Os moléculas de RNA brotam do DNA 
•  Os RNA são produzidos a partir de 
regiões discretas, os cistrons 
Transcrição x Replicação 
Aspectos Comuns: 
•  Polaridade 5’ → 3’; 
•  Necessidade de um molde; 
 
Aspectos Diferenciais: 
•  Não requer “primer” (iniciador) 
•  Apenas um segmento de DNA é Transcrito 
•  Uma das fitas de DNA serve como molde 
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A RNA polimerase acrescenta 
nucleotídeos à porção 3´ 
•  (NMP)n + NTP → (NMP)n+1 + PPi PPi → 2 Pi 
Unidade Transcricional 
•  A síntese do RNA começa e termina em regiões 
específicas do genoma 
•  Os promotores demarcam o início da transcrição 
•  Os terminadores demarcam o final do processo 
Região codificadora (quando houver) 
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O RNA é sintetizado na direção 5´ 
para o 3´ 
•  Por definição a fita sintetizada, o transcrito primário, é idêntica 
à fita codificadora (exceto T->U) ... 
•  e complementar à fita molde, que é o substrato da RNA pol 
        5' ACTGCTGACTGATCAG 3' DNA fita codificadora ou senso 
        3‘ TGACGACTGACTAGTC 5' DNA fita molde ou antisenso 
 
 
        5' ACUGCUGACUGAUCAG 3' RNA transcrito 
  
Fita codificadora ou fita senso: sequência “idêntica” a do mRNA (T➠U) 
Fita ou molde ou antisenso: sequência complementar a do mRNA 
O Processo de transcrição 
Fita molde 
Fita codificadora 
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Micrografia mostrando RNAs sendo produzidos 
a partir de duas regiões codificadoras 
A RNA polimerase 
A RNA Polimerase não 
Precisa de iniciador para 
Começar a síntese do 
transcrito 
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Evolução da RNA polimerase 
Figure 6-10 Molecular Biology of the Cell (© Garland Science 2008) 
As RNA polimerases tem 
origem filogenética comum 
The structures of RNA polymerases (RNAPs) in bacteria (Thermus aquaticus; Protein Data Bank (PDB) accession 1I6V), archaea (Sulfolobus 
solfataricus; PDB accession 2PMZ) and eukaryotes (Saccharomyces cerevisiae RNAP II; PDB accession 1NT9). Homologous subunits are 
colour-coded and clearly demonstrate the high degree of similarity between all three transcription engines and, in particular, how the archaeal 
RNAP closely resembles eukaryotic RNAP II. 
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Metabolismo informacional de Archaeas 
é mais próximo de eucariotos 
Genero Sulfolobus 
http://web.pdx.edu/~kstedman/research.html 
RNA Polimerase Eucariótica 
•  Eucariotes tem mais de uma RNA polimerase 
RNA polimerase eucarióticas 
Enzima Localização Produto Atividade Relativa 
Sensibilidade à 
α-amanitina 
 
RNA pol I Nucléolo rRNA 50-70% Insensível 
RNA pol II Nuceloplasma mRNA 20-40% Sensível 
RNA pol III Nuceloplasma tRNA ~10% Pouco sensível 
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A transcrição 
ocorre em etapas 
•  Iniciação 
•  Elongação 
•  Terminação 
Cada gene produz quantidades 
distintas de RNA e proteína associada 
Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008) 
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Como medir a abundância de 
um transcrito? 
•  Métodos específicos 
–  Northern blot 
–  PCR em tempo real 
•  Métodos globais 
–  Micro-arranjos 
–  RNA-seq 
Northern blot 
http://www.discoveryandinnovation.com/BIOL202/notes/lecture26.html 
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Hibridização em 
micro-arranjos 
Análise de micro-arranjos 
 
http://www.discoveryandinnovation.com/BIOL202/notes/lecture26.html 
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Chip de alta densidade 
RNA-seq 
http://www.genomics.hk/Transcriptome.htm 
² O sequenciamento do conjunto de transcritos e seu mapeamento 
em um genoma de referência revela as regiões no genoma que 
estão sendo ativamente transcritas 
Kelly et al, 2014 
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Cluster analysis – visão global 
do transcritoma 
http://www.discoveryandinnovation.com/BIOL202/notes/lecture26.html 
Rede regulatória 
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O Piano e o Partitura 
Não basta um piano, é necessário 
uma programação, a partitura, 
para se tocar uma melodia 
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A expressão de um gene pode 
ser regulada em diversos níveis 
1.  Controle da transcrição 
2.  Controle do Processamento do RNA 
3.  Controle da localização e transporte do RNA 
4.  Controle da tradução 
5.  Controle da degradação do RNA 
6.  Controle da atividade proteica 
Bases da regulação 
 Interação proteínas-Ácidos 
nucléicos 
http://www.grs-sim.de/research/computational-biophysics/research-activity/hiv-1.html 
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Observando a interação DNA-Proteína 
Gel de retardo 
Gel shift assay