Beruflich Dokumente
Kultur Dokumente
Traduo
Traduo: processo que leva a converso da sequncia de nucletidos do mRNA em sequncia de aminocidos de pptido
Traduo
aa8-tRNA entrando
AGC
mRNA
GUC UGG AG U
UCG
Traduo: RNA-> protena Adio de um aminocido cadeia polipeptdica crescente durante a traduo do mRNA
tRNA
-70-80 nucletidos -alguns nucletidos no tRNA so modificados, como di-hidrouridina (D), pseudouridina (Y), inosina (I). Di-hidrouridina-H adicionada a C5 e C6 do uracilo Pseudouridina-ribose ligada ao C5 e no N1. Alguns nuclesidos so metilados
Estrutura secundria do tRNA Azul-nucletidos modificados m-metilado Anticodo-trinucletido complementar codo do mRNA
ao
Transferncia tRNA
tRNA molecules share many structural similarities but each has a unique three-dimensional shape that allows recognition by the correct synthetase which catalyzes the joining of a tRNA with its specific amino acid to form an aminoacyl-tRNA
mRNA
Wobbling
Traduo=>Emparelhamento entre anticodo do tRNA e codo 61 codes Se cada codo emparelhasse com um s anticodo, haveria 61 tRNAs Bactrias 30-40 tRNAs com diferentes anticodes
mRNA
Wobbling
Emparelhamentos possveis na posio wobble Uma base pode emparelhar com vrias bases.
Emparelhamentos no standard
Cdigo gentico
Sntese proteica Relao entre sequncia de nucletidos de mRNA e sequncia de aminocidos de protenaCdigo gentico (Nirenberg, anos 60)
Cdigo gentico standard Codo tripleto no mRNA
Sntese de pptido inicia-se sempre com aminocido metionina (Met)- AUG. Codo stop (UAA, UAG ou UGA) significa terminar pptido. ORF- Open Reading Frame sequncia de DNA desde codo de iniciao (ATG) at codo stop (TAA, TAG ou TGA) que provavelmente (mas no necessariamente) codifica polipptido.
Cdigo Gentico
O cdigo gentico :
-degenerado- um aminocido pode ser codificado por vrios codes
wobble pairing 3 posio do codo mais flexvel t ...G t ...U m...C m ...A U G
Aminocidos
Prmio Nobel Prize in Physiology or Medicine 1968 Nirenberg, Khorana e Holley "for their interpretation of the genetic code and its function in protein synthesis"
http://nobelprize.org/educational_games/medicine/gene-code/history.html
-Induzidas (mutagnese) -Elementos infecciosos (vrus como herpes simplex, etc) podem causar cortes e deleces pois libertam nucleases Consequncias das mutaes: -alterao de sequncia de protena -alteraes na regulao de produto de gene (controlo da expresso gentica)
Mutaes
Silenciosa
Neutra
Missense
Nonsense
Frameshift
http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c
http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c
Homo sapiens UUU 17.1 UCU 14.7 UUC 20.6 UCC 17.6 UUA 7.5 UCA 12.0 UUG 12.6 UCG 4.4 CUU 13.0 CUC 19.8 CUA 7.8 CUG 39.8 AUU 16.1 AUC 21.6 AUA 7.7 AUG 22.2 CCU 17.3 CCC 20.1 CCA 16.7 CCG 6.9 ACU 13.0 ACC 19.4 ACA 15.1 ACG 6.1
UAU 12.1 UAC 15.5 UAA 0.7 UAG 0.6 CAU CAC CAA CAG AAU AAC AAA AAG 10.5 15.0 12.0 34.1 16.7 19.5 24.1 32.2
UGU 10.0 UGC 12.2 UGA 1.5 UGG 12.7 CGU 4.6 CGC 10.7 CGA 6.3 CGG 11.6 AGU AGC AGA AGG 11.9 19.3 11.5 11.4
Cdigo gentico
Aminoacil-tRNA
Aminoacil-tRNA: aminocido ligado ao extremo 3 do tRNA enzimas sintetases de aminoacil-tRNA tRNA+aminocido -> tRNA carregado com aminocido
Exemplo: tRNAArg + Arg -> Arg-tRNAArg Apenas um aminocido especfico pode ser ligado ao extremo 3' de determinado tRNAenzimas reconhecem anticodo do tRNA e o aminocido compatvel 20 sintetases de aminoacil-tRNA diferentes (cada uma adiciona apenas um dos 20 aminocidos a um tRNA compatvel)
Formao de ribossoma
1-Sntese de molculas de RNA 2-Transporte para citoplasma (eucariotas) 3-Associao com protenas ribossomais (Ls e Ss)
Traduo
http://nobelprize.org/educational_games/medicine/dna/a/translation/index.html
rRNA 16S do ribossoma contm sequncia que emparelha com sequncia Shine-Dalgarno
5'--UAAGGAGG(5-10 bases) AUG 3'--AUUCCUCC........ 16S rRNA mRNA
Iniciao da traduo
Como reconhecido o codo de iniciao AUG correcto?
Met - Arg-...
Traduo
Iniciao
Incio metionina (Met) Aminoacil-tRNA inicial Met-tRNAiMet Bactrias Formilmetionina (fMet)= Metionina do aminoaciltRNA inicial modificada pela adio de grupo formil (HCO) no grupo amino. fMet composto estranho a eucariotas resposta imune Humanos resposta imune a pptido "fMet-Leu-Phe" 1000 vezes maior que resposta imune a "Met-LeuPhe"
http://nobelprize.org/educational_games/medicine/dna/a/translation/initiation.html
Elongao
Dois locais de ligao ao tRNA: local A (aminoacil) e local P (peptidil) a-Met-tRNAiMet inicial entra no local P Nova entrada Novo aminoacil-tRNA entra no local A.
Passo catalizado por factores de elongao (Tu e Ts em procariotas e por EF1 e EF1b em eucariotas)
b-Metionina transferida para novo formando "peptidil-tRNA" aminoacil-tRNA,
c-Pptido ligado ao Peptidil-tRNA no local P transferido para novo aminoacil-tRNA no local A-passo catalizado por Peptidil transferase d-Peptidil-tRNA gerado no local A ocupa local P vazio-catalizado por factor de elongao G bactrias e factor de elongao EF eucariotas e-tRNA no local P ejectado do ribossoma f-Ribossoma move-se um codo na cadeia do mRNA
Terminao
Quando ribossoma pra em codo stop Protenas- Factores de terminao que reconhecem codes stop-estimulam libertao de peptidiltRNA do ribossoma. Depois peptidil-tRNA separa-se em tRNA e nova cadeia peptdica sintetizada.
Sem mRNA, Ribossoma separa-se em subunidade maior e menor.
Modificaes ps-traducionais
Folding
Chaperoninas-Protenas que ajudam as protenas a adquirirem estrutura correcta
Prevenir folding incorrecto de protena pela chaperonina BiP (a) Antes de peptdo estar completamente sintetizado pode enrolar permaturamente pois regies hidrofbicas tendem a agregar
(b) BiP pode ligar-se a regies hidrofbicas, prevenindo folding incorrecto prematuro
Folding
Protenas sintetizadas no citoplasma - transportadas para locais apropriados na clula -Sequncias sinal especficas- direccionam protenas
KDEL -"Lys-Asp-Glu-Leu"
Glicosilao (Aparelho de Golgi) adio de manose 6-fosfato (M6P)
Protenas Estrutura
Proteasoma- grande complexo, contendo protenas com actividade de ligao a ubiquitina, protease, etc
Jusante ( Downstream)- direco da transcrio Montante (upstream)- oposto da direco de transcrio "+1" Incio da transcrio (no h base 0: base a montante de +1 1)
Muitos promotores no tm TATA mas apenas o elemento iniciador (INR) funcionalmente anlogo. BRE, TFIIB recognition element; DPE, downstream promoter element; CTF, CCAAT-binding transcription factor; CBF, CCAAT box-binding factor; TBP, TATA box-binding protein; TAF, TBP-associated factors.
Promotores
regio do DNA de iniciao da transcrio-reconhecida por factores de transcrio
Lactose->glucose+galactose
Ligao da RNA polimerase ao promotor lac fraca -Transcrio requer frequentemente activao ->catabolite activator protein (CAP)
Silenciador (ou operator) do opero lac- localizado no local de incio da transcrio.
Promotor do opero lac Sequncia de consenso do s70 (regula opero lac) TATAAT em -10 e TTGACA em 35. Sequncia 10 TATGTT difere em dois nucletidos Sequncia 35 TTTACA difere em um nucletido.
a curta distncia do extremo 5 de gene, que actua como local de ligao da RNA polimerase ou factores de transcrio.
Enhancers
Elementos de regulao positivos localizados a montante ou jusante do local de incio da transcrio (maioria a montante). Procariotas- prximos do promoter Eucariotas-podem estar longe do promotor. Regio enhancer pode conter um ou mais elementos reconhecidos por activadores de transcrio. Exemplos
-Gene
glnA de E. coli
Enhancer tem dois locais de ligao ao factor de transcrio nitrogen regulatory protein C (NTRC).
Genes GAL1 e GAL10, transcritos em direces opostas, so regulados pelo mesmo enhancer entre eles. upstream activating sequences (UAS), com 4 locais de ligao ao factor de transcrio GAL4.
Controle da transcrio por regio enhancer (HS1 - HS4) que contm locais de ligao a activadores de transcrio como GATA-1, NF-E2, AP-1. Locus Control Region (LCR)-regula expresso dos 5 genes (e, Gg, Ag, d e b)
Silenciadores
Elementos de DNA que interagem com repressores (protenas) inibindo a transcrio Procariotas Exemplos: Procariotas Operadores do Opero lac e do opero trp. Eucariotas Silenciadores dos genes: >Gene da b globina humano >Gene CD95(Fas/APO-1) humano >Gene dopamine beta-hydroxylase (DBH) humano Silenciadores so conhecidos como operadores
Por vezes elemento de DNA pode actuar como enhancer ou silenciador, dependendo da protena de ligao. Alguns genes tm box E (consenso CACGTG) -liga dmero Max/Myc- activa transcrio -liga dmero Max/Mad suprime transcrio
Elementos de Resposta
Locais de reconhecimentos de certos factores de transcrio Maioria localizados a menos de 1 kb do incio da transcrio. Elementos de Resposta de eucariotas
Response Element CRE ERE GRE HSE SRE Transcription Factor CREB Estrogen receptor Glucocorticoid receptor Heat shock factor Serum response factor Consensus Sequence TGACGTCA AGGTCANNNTGACCT AGAACANNNTGTTCT GAANNTTCNNGAA CC(A/T)6GG
Elemento de resposta do cAMP (CRE) interage com CREB (CRE-binding protein) Elemento de resposta do estrognio (ERE) e elemento de resposta do glucocorticide (GRE) so locais de reconhecimento do receptor do estrognio e do receptor do glucocorticide, respectivamente. Elemento de resposta de choque trmico (HSE) encontra-se em genes de protenas de choque trmico. Serum response element (SRE) liga-se ao serum response factor (SRF) que pode ser activado por muitos factores de crescimento no soro.
Factores de Transcrio
Protenas que tm motif especfico para interagirem com o DNA no incio da transcrio Motifs mais comuns: >Zinc finger >Helix-turn-helix >Leucine zipper >Helix-loop-helix
Leucine Zipper
Factores de transcrio com leucine-zipper ligam DNA como dmeros. leucine zipper formado por duas hlices a, uma de cada monmero
Factores de Transcrio
Estrutura de regio zinc finger
Interaco entre dmero de repressor l e DNA Cada repressor l contm uma regio helix-turn-helix
Ligao Protenas-DNA
AP-1
TGAGTCA
p53
PuGPuCATGPyCPy
AP-2
CCC(A/C)N(C/G)3
NF-kB
GGGPuNTPyPyCC
Oct-1
ATGCAAAT
NFAT
GGAGAPu
GATA-1
(A/T)GATAPu
NF-E2
TGACTCAG
Prmio Nobel em Fisiologia ou Medicina 2006 "for their discovery of RNA interference - gene silencing by double-stranded RNA"
Andrew Z. Fire
Craig C. Mello
http://nobelprize.org/nobel_prizes/medicine/laureates/2006/adv.html
O complexo RISC (RNA-induced silencing complex), liga-se ao mRNA, por emparelhamento com o pequeno RNA antisense do siRNA
Nalguns sistemas, em particular plantas, vermes e fungos, uma RNA dependent RNA polymerase (RdRP) gera e/ou amplifica siRNA
Implicaes do RNAi
1. RNAi protege contra infeces virais 2. RNAi garante estabilidade do genoma mantendo silenciosos os elementos mveis 3. Mecanismos tipo RNAi reprimem sntese proteca e regulam o desenvolvimento de organismos-MicroRNAs 4. Mecanismos tipo RNAi mantm a cromatina condensada e suprimem a transcrio 5. RNAi representa uma nova ferramenta experimental para reprimir gene especfico 6. RNAi pode ser uma abordagem til em terapia gentica futura
Esquema hipottico de como combinaes de algumas protenas reguladoras de genes podem originar muitos tipos diferentes de clulas durante desenvolvimento.
PrmioNobel em Fisiologia ou Medicina 2002 "for their discoveries concerning 'genetic regulation of organ development and programmed cell death'"
Sydney Brenner
H. Robert Horvitz
John E. Sulston By using the nematode Caenorhabditis elegans as a model system, the Laureates have identified key genes regulating these processes. They have also shown that corresponding genes exist in higher species, including man.
http://nobelprize.org/nobel_prizes/medicine/laureates/2002/illpres/
The diagram shows some of the gene regulatory proteins thought to control expression of this gene during red blood cell development. Some of the gene regulatory proteins shown, such as CP1, are found in many types of cells, while others, such as GATA-1, are present in only a few types of cells, including red blood cell precursors, and are therefore thought to contribute to the cell-type specificity of betaglobin gene expression. As indicated by the bidirectional arrows, several of the binding sites for GATA-1 overlap those of other gene regulatory proteins; it is thought that occupancy of these sites by GATA-1 excludes binding of other proteins. (Adapted from B. Emerson, In Gene Expression: General and Cell-Type Specific (M. Karin, ed.), pp. 116-161. Boston: Birkhauser, 1993.)
1. RNAi protege contra infeces virais: The finding of Fire and Mello that cells can process injected dsRNA and eliminate homologous single-stranded RNA suggested that RNAi could constitute a defence mechanism against viral attacks. It had earlier been shown that plant cells have an efficient defence against viruses based on the PTGS phenomenon40,41. When it became apparent that PTGS is the plant equivalent to RNAi, this early work in plants supported the proposition that RNAi is involved in protecting cells from viral attacks. Today, we know that this anti-viral mechanism is at work in plants, worms and flies, whereas it is still unclear how relevant it is for vertebrates, including man. 2. RNAi garante estabilidade do genoma mantendo elementos mveis silenciosos: It was proposed early on that RNAi/PTGS in C. elegans and plants could block the action of transposons (mobile elements in the genome). Subsequently, it could be shown that when components of the RNAi machinery are mutated in C. elegans, transposons are activated and the mobile elements cause disturbances in the function of the genome42,43. It has been proposed that in transposon-containing regions of the genome both DNA strands are transcribed, dsRNA is formed, and the RNAi process eliminates these undesirable products. As short dsRNAs can also operate directly on chromatin and suppress transcription (see below), this would be another mode to keep transposons inactive (see below under 4). Even if the mechanisms are not yet fully revealed, it is clear that if the RNAi machinery is not efficient, the transposons are not kept under control and can start to jump and cause deleterious effects in the genome. It has been argued that RNA silencing could represent an "immune defence" of the genome44. Close to 50% of our genome consists of viral and transposon elements that have invaded the genome in the course of evolution. The RNAi machinery can recognize invading double-stranded viral RNA (or the double-stranded replicative form of the viral RNA) and suppress the infection by degradation of the RNA. The RNAi system thus shares important features with the vertebrate immune system: it recognizes the invading parasite (dsRNA), raises an initial response and subsequently amplifies the response to eliminate the foreign element.
3. Mecanismos tipo RNAi reprimem sntese proteca e regulam o desenvolvimento de organismos : Soon after the discovery that short RNA is the effector of RNAi, it was shown that there is a class of endogenous RNA molecules of the same size in worms, flies, mice and humans; this small RNA was called microRNA (miRNA)8-10. Plants also contain this class of endogenous RNA45. The revelation of miRNA led to intense research on the nature of these RNA molecules. The C. elegans lin-4 (ref. 5) and let-7 (ref. 7) RNAs were regarded as prototypes, and examples of similar cases were soon revealed in several organisms46. The small miRNAs are processed from larger hairpin-like precursors by an RNAi-like machinery47,48 (Fig. 4). The miRNAs can regulate gene expression by base-pairing to mRNA, which results in either degradation of the mRNA or suppression of translation. Today, it is estimated that there are about 500 miRNAs in mammalian cells, and that about 30% of all genes are regulated by miRNAs. It is known that miRNAs play an important role during development in plants, C. elegans and mammals. Thus, the miRNA-dependent control of gene expression represents a new major principle of gene regulation. However, the full significance of small regulatory RNAs is probably still not apparent. 4. Mecanismos tipo RNAi mantm a cromatina condensada e suprimem a transcrio: It was known from work in plants that gene silencing could take place at the transcriptional level (TGS). After the discovery of RNAi, it was soon shown that TGS in plants operates via RNAi-like mechanisms49,50. In the fission yeast Schizosaccharomyces pombe51,52, and later on in Drosophila and vertebrates, it was found that similar processes keep heterochromatic regions condensed and transcriptionally suppressed. In addition, the RNAilike machinery regulates the activity of genes in the immediate vicinity of the condensed blocks of chromatin. The phenomenon is still not understood at the molecular level although histone modifications, binding of specific chromatin condensing proteins (HP1), and DNA methylation all play important roles46. It is, however, evident that this action on chromatin is most important for proper functioning of the genome and for maintenance of genome integrity. 5. RNAi reperesenta uma nova ferramenta experimental para reprimir gene especfico. The targeted action of RNAi immediately suggested that this phenomenon could be utilized as a general method to suppress specific genes and look for the resulting phenotypic effect. It was also soon evident that this could be accomplished in such an efficient manner that essentially any gene in an organism could be studied functionally. After the initial work in C. elegans, this technical approach could be applied to cells from almost all organisms, including mammalian cells. This targeted gene silencing by RNAi has already had a tremendous impact in studies of the function of individual genes. It is now exploited not only in cultured cells but also in transgenic organisms. DNA constructs are introduced into the organisms under appropriate promoter control, and dsRNA hairpin structures are produced and further processed to achieve specific effects on gene regulation. 6. RNAi pode ser uma abordagem til em terapia gentica futura The possibility to achieve RNAi-governed gene regulation in transgenic organisms has stimulated many explorations of whether this would be a useful option for medical therapy53,54. Promising results have been reported in several animal models55-58 and even in recent clinical trials, but it is too early to predict the outcome of these challenging efforts.