Tradução I - coltri.bio.br · Tradução: processo pelo qual a sequência de nucleoAdeos de um...

TraduçãoI

T.thermophilusribossomo70S

ü RNAs

ü códigogené5co

ü Traduçãoemprocariotos

ü Traduçãoemeucariotos

Roteirodaaula

Tradução:processopeloqualasequênciadenucleoAdeosdeummRNAéusadacomomoldepara

criarumacadeiapolipepAdica

Lodisch,7thed

RNAsnatradução

In eukaryotic DNA, each protein-coding gene is transcribed from its own promoter. The initial primary transcript very often contains noncoding regions (introns) interspersed among coding regions (exons). • Eukaryotic primary transcripts must undergo RNA pro-cessing to yield functional RNAs. During processing, the ends of nearly all primary transcripts from protein-coding genes are modified by addition of a 5' cap and 3' poly(A) tail. Transcripts from genes containing introns undergo splicing, the removal of the introns and joining of the exons (see Figure 4-15). • The individual domains of multidomain proteins found in higher eukaryotes are often encoded by individual exons or a small number of exons. Distinct isoforms of such proteins often are expressed in specific cell types as the result of alter-native splicing of exons.

4.3 The Decoding of mRNA by tRNAs Although DNA stores the information for protein synthesis and mRNA conveys the instructions encoded in DNA, most biological activities are carried out by proteins. As we saw in Chapter 3, the linear order of amino acids in each protein determines its three-dimensional structure and activity. For this reason, assembly of amino acids in their correct order, as encoded in DNA, is critical to production of functional pro-teins and hence the proper functioning of cells and organisms.

Translation is the whole process by which the nucleotide sequence of an mRNA is used as a template to join the amino acids in a polypeptide chain in the correct order (see Figure 4-1, step D ). In eukaryotic cells, protein synthesis occurs in the cytoplasm, where three types of RNA mole-cules come together to perform different but cooperative functions (Figure 4-17):

1. Messenger RNA (mRNA) carries the genetic informa-tion transcribed from DNA in a linear form. The mRNA is read in sets of three-nucleotide sequences, called codons, each of which specifies a particular amino acid.

2. Transfer RNA (tRNA) is the key to deciphering the codons in mRNA. Each type of amino acid has its own subset of tRNAs, which bind the amino acid and carry it to the growing end of a polypeptide chain when the next codon in the mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains a three-nucleotide se-quence, an anticodon, that can base-pair with its comple-mentary codon in the mRNA.

3. Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes. These complex structures, which physi-cally move along an mRNA molecule, catalyze the assem-bly of amino acids into polypeptide chains. They also bind

tRNA4 leaving

mANA

aa1

aartRNA7 arriving 1;1

H2N- c - R 7

C==o I

0

Movement of ribosome

FIGURE 4 -17 The three roles of RNA in protein synthesis. Messenger RNA (mRNA) is translated into protein by the joint action of transfer RNA (tRNA) and the ribosome, which is composed of numerous proteins and three (bacterial) or four (eukaryotic) ribosomal RNA (rRNA) molecules (not shown). Note the base pairing between tRNA anticodons and complementary codons in the mRNA. Formation of a peptide bond between the amino-group N on the incoming aa-tRNA and the carboxy-terminal Con the growing. protein chain (green) is catalyzed by one of the rRNAs. a a = amino acid; R - side group. [Adapted from A. J. F. Griffiths et al., 1999, Modern Genetic Analysis, W. H. Freeman and Company.]

tRNAs and various accessory proteins necessary for protein synthesis. Ribosomes are composed of a large and a small subunit, each of which contains its own rRNA molecule or molecules.

These three types of RNA participate in the synthesis of proteins in all organisms. In this section, we focus on the de-coding of mRNA by tRNA adaptors, and how the structure of each of these RNAs relates to its specific task. How they work together with rRNA, ribosomes, and other protein factors to synthesize proteins is detailed in the following section. Because translation is essential for protein synthesis, the two processes commonly are referred to interchangeably. However, the polypeptide chains resulting from translation undergo post-translational folding and often other changes (e.g., chemical modifications, association with other chains) that are required for production of mature, functional proteins (Chapter 3).

Messenger RNA Carries Information from DNA in a Three-letter Genetic Code As noted above, the genetic code used by cells is a triplet code, with every three-nucleotide sequence, or codon, being "read" from a specified starting point in the mRNA. Of the 64 possible codons in the genetic code, 61 specify individual amino acids and three are stop codons. Table 4-1 shows that most amino acids are encoded by more than one codon.

4.3 The Decoding of mRNA by tRNAs 131

ü  RNAsmensageiros:nucleoAdeosem“codons”

ü  RNAsdetransferência(transportadores):an5codons,carregamaa

ü  RNAribossomal:formamosribossomos

Lodisch,7thed

mRNAs

ü  Processamentoco-transcricionalü  Exportaçãoü  ProteínasEJC/CAP5’/poly-A:interaçãocomfatoresdeiniciaçãodatradução

ProcessamentodetRNA

ü splicing(intronazul)

ü modificaçãodebases

Lodisch,7thed

tRNAs:osadaptadores

Alberts,5thed

tRNAs:osadaptadores

estruturasecundáriatRNAscarregadoscomaa

Alberts,5thed

tRNAs:osadaptadores

•  aminoacil-tRNAsynthetase(específicas):adiçãodoaaaotRNAcorrespondente(comgastodeATP)

•  500genestRNAsemhumanos,somente48an5codons• maisdeumtRNAporaa/tRNAsreconhecemmaisdeumcodon

Alberts,5thed

tRNAs:osadaptadores

aa-tRNAsynthetasescorrigeminserçãodeaaerrado

Alberts,5thed

rRNAs

Alberts,5thed

rRNAs

M. Fromont-Racine et al. / Gene 313 (2003) 17–42 29

Fromont-Racineetal,Gene,2003

ü  rRNAssãoprocessados

ü Montagemdesubunidadescomeçanonúcleo

Ribossomos

Alberts,5thed

Ribossomos

Ribossomos:ribozimas

Alberts,5thed

Estruturacristalográficadoribossomo

Ada Yonath Thomas Steitz Venkatraman Ramakrishnan

Harry Noller

RibossomosdebactériaseanDbióDcos

© 2005 Nature Publishing Group

Spectinomycin

Tetracycline 1

Pactamycin

Hygromycin B

Streptomycin

Paromomycin,geneticin

Tetracycline 2

Thiostrepton

Avilamycin

Streptogramin A,chloramphenicol,

puromycin

Pleuromutilins

Streptogramin B,lincosamides

Macrolides

Antibiotic-binding sites on the ribosomeGiven the fundamental importance of the rRNA, it is not surprising that most ribosome inhibitors target the rRNA-rich surfaces on the 30S and 50S subunits (FIG. 1). The crystal structures of many ribosome-targeting anti-biotics have been solved in complex with their ribosomal sub unit. The 30S subunit is targeted by drugs that include tetra cycline, pactamycin and the amino glycosides

see TABLE 2, which hinder the subunit in carrying out its principle function of deciphering the genetic informa-tion encoded in the mRNA BOX 1. Binding of amino-glycosides such as geneticin, paromomycin (FIG. 2) and apramycin at the decoding site of the 30S subunit have been well studied by NMR44,45 and crystallography46–49.

The 50S subunit is targeted by a wide range of drugs that bind within three main regions (FIG. 1) to inter-fere with the subunit’s main functions in controlling GTP hydrolysis, the formation of peptide bonds, and channelling the peptide through the subunit tunnel BOX 1. Binding of the thiopeptide antibiotics such as thiostrepton inhibit GTP-associated processes50–52, whereas the oligosaccharide antibiotics avilamycin and evernimicin interrupt a subset of these processes by binding to their own distinct site53–56. So far, there are no crystallographic data on the thiopeptide or oligo-saccharide compounds bound to their sites on the 50S subunit. Interactions of drugs at the third bind-ing region on the 50S subunit (FIG. 1) have, however, been subject to rigorous crystallographic study. This latter region is extensive, covering the upper part of the tunnel together with the peptidyl-transferase centre, and accommodates a diverse range of drugs, including the MLSB (macrolide, lincosamide, streptogramin B) compounds1,2,42,57–60, chloramphenicol57,59, puromycin16, pleuromutilins61,62 and oxazolidinones63,64.

Below, we concentrate on drug targets at the two main and best-characterized reaction centres in the ribosome — the aminoglycoside target at the decod-ing site on the 30S subunit, and the target for MLSB compounds adjacent to the peptidyl-transferase centre in the 50S subunit.

The decoding site on the 30S subunitThe decoding site is part of the ribosomal A site and is situated at the end of the 16S rRNA helix 44 on the 30S subunit interface BOX 1. The function of the decoding site is to monitor codon–anticodon pairing after the aminoacylated tRNA has been placed in the

Figure 1 | Binding sites of antibiotics on the bacterial ribosome. The 30S ribosomal subunit is shown on the left and the 50S ribosomal subunit is shown on the right. The antibiotic-binding sites were initially determined by biochemical and genetic techniques; subsequently, many sites were revealed in greater detail by X-ray crystallography. At the overlapping sites, antibiotic binding is usually mutually exclusive (for example, for macrolide, lincosamide and streptogramin B compounds), however, streptogramin A and B compounds bind synergistically at adjacent sites. Subunit models are based on the Thermus thermophilus 70S ribosome structure29. In this figure, for clarity, part of the r-protein L9 has been omitted. Ribosomal RNAs are shown in yellow and grey and r-proteins in bronze and blue.

Table 2 | Antibiotics that target the 30S ribosomal subunit

Drug PDB Resolution (Å) System Ref.

Aminoglycosides*

Streptomycin 1FJG 3.0 Thermus 46

Paromomycin 1FJG 3.0 Thermus 46

Hygromycin B 1HNZ 3.3 Thermus 119

Paromomycin 1IBK 3.3 Thermus 66

Paromomycin 1J7T 2.5 RNA fragment 47

Tobramycin 1LC4 2.5 RNA fragment 120

Geneticin 1MWL 2.4 RNA fragment 48

Apramycin 1YRJ 2.7 RNA fragment 121

Tetracyclines‡

Tetracycline 1HNW 3.4 Thermus 119

Tetracycline 1I97 4.5 Thermus 122

Cyclic peptides‡

Viomycin - - nd -

Capreomycin - - nd -

Other 30S drugs§

Edeine 1I95 4.5 Thermus 122

Spectinomycin 1FJG 3.0 Thermus 46

Pactamycin 1HNX 3.4 Thermus 119*Bind to aminoacyl or peptidyl sites and induce errors in translation. ‡Block binding of transfer RNA to aminoacyl site. §Various effects, including inhibition of translocation. The Thermus system refers to 30S subunits from the bacterium Thermus thermophilus; RNA fragment contains the decoding region of 16S rRNA. nd, crystal structure not determined; PDB, Protein Data Bank ID.

NATURE REVIEWS | MICROBIOLOGY VOLUME 3 | NOVEMBER 2005 | 873

R E V I E W S

Poehlsgaard&Douthwaite,NatRevMicro2005

RibossomosdebactériaseanDbióDcos

Alberts,5thed

CódigogenéDco

•  universal•  codons:triosde

nucleoAdeos

•  4basesdiferentes:4x4x4=64combinaçõespossíveis

•  somente20aminoácidos-mesmocodoncodificaaadiferentes:redundância

CódigogenéDco

• Degenerado•  3ºnucleoAdeo:pareamento

difereepodeocorrercombasesmodificadas

•  Conformaçãodoan5codon

permitemaiorflexibilidade:Wobblehypothesis

CódigogenéDco

Wobblehypothesis

CódigogenéDco

• codonsespecíficos• codonusage/codonbias

Lodisch,7thed

Pautadeleitura

THE FAT CAT ATE THE RAT

HEF ATC ATA TET HER AT

Pautadeleituracorreta

Pautadeleituraerrada:mensagemerrada!

Pautadeleitura

• sequênciadocodoninicialatécodondeparada:pautadeleitura(“readingframe”)

• 3possíveispautasdeleitura-geralmentesomente1geraproteínafuncional

Lodisch,7thed

Pautadeleitura

Mecanismodetradução

ExitPep5dil-transfer Acceptor

Eucariotos:2aa/segProcariotos:20aa/seg

Alberts,5thed

Tradução EF-Tu/EF-G– bactériasEF1/EF2-eucariotos

Alberts,5thed

Tradução

•  Proofreading

•  InteraçõescomRNAsribossomaisasseguraminserçãoaacorreto

•  99,9%precisão

Alberts,5thed

Tradução

Traduçãoemprocariotos

•  RBS:sequência“Shine-Dalgarno”-5’AGGAGGU3’

•  Pareamentocom16SrRNAparaposicionaroribossomocorretamente(+outrosfatores)

Alberts,5thed

Iniciaçãodatradução(procariotos)

Controledatraduçãoemprocariotos

Alberts,5thed

eiF4 complex {

@

5 .• _5? ____ _

FIGURE 4-24 Initiation of translation in eukaryotes. The current model of eukaryotic initiation involves eight steps. Step 0: An eiF2 ternary complex forms when eiF2-GTP binds a tRNA,M••. Step fJ: When a ribosome dissociates at the termination of translation, the 40S subunit is bound by eiFl, eiFl A, and eiF3. A 43S preinitiation complex forms when this associates with an eiF2 ternary complex and e1F5. Step 10: An mRNA is activated when a multiple-subunit eiF4 complex binds: subunit eiF4E binds to the 5' cap structure and subunit eiF4G binds multiple copies of the poly(A)-binding protein (PABP) bound to the mRNA poly(A) tail. Subunit e1F4A RNA helicase activity unwinds any RNA secondary structure at the 5' end of the mRNA. eiF4B, which stimulates helicase activity, also joins this circular complex in which both the mRNA 5' cap and poly(A) tail are associated w ith the e1F4 complex. Step D: The 43S preinitiation complex binds an eiF4-mRNA complex. Step r!l: The eiF4A RNA helicase unwinds RNA secondary structure as the 40S complex scans in the direction until it recognizes the initiation codon. Step rl'!: Recognition of the initiation codon causes eiF5 to stimulate hydrolysis of eiF2-bound GTP. This switches the conformation of the scanning complex to a 48S initiation complex with the anticodon oftRNA,Met base-paired to the initiator AUG in the 40S P site. Step 6 : The 60S subunit joins the 40S subunit, leading to release of most the earlier-acting eiFs as eiF5B-GTP binds to eiF1A in the ribosomal A site. The released e1F4 complex and e1F4B associate with the cap and PABP as shown in step 1D to prepare for interaction with another 43S preinitiation complex. For simplicity, this is not shown. Step(;): Correct association of the 40S and 60S subunit results in hydrolysis of eiF5B-bound GTP, release of eiF5B-GDP and eiFl A, and formation of the 80S initiation complex with tRNA,M••base-paired to the initiation codon in the ribosomal P site [Adapted from R. J. Jackson et al .. 201 o, Nar. Rev. Mol. Cell Bioi. 1 0:113.)

Met e1F2 ternary complex Met

formation

D >>----+ @GTP

405

(?}GTP:r-435 complex format1on

0 }

2 GTP 5 435 preinitiation

Met

complex

Attachment to mRNA

AUG

5' to 3' scanning

Initiation codon recognition, hydrolysis of e1F2-bound GTP and P, release

} 485 Initiation complex

Hydrolysis of eiF5B-bound GTP and release of eiF5B and eiF1A

® ®GOP

80S Initiation complex

4.4 Stepwise Synthesis of Proteins on Ribosomes 139

Iniciaçãodatradução(eucariotos)

•  eukaryo5cIni5a5onFactors– eIF

•  ComplexoeIF4:associaçãoaomRNA

•  ComplexoeIF2:complexoternárioeIF2-GTP+tRNAMet

•  SomentetRNA-Metpodeseligardiretamenteaosí5oP

Lodisch,7thed

eiF4 complex {

@

5 .• _5? ____ _



formation

D >>----+ @GTP

405


0 }


Met

complex

Attachment to mRNA

AUG

5' to 3' scanning




® ®GOP




Scanning5’-3’

•  eIF4E/eIF4G:reconhecemmRNA(eCAP)ejuntocomsubunidademenor“escaneiam”mRNAembuscadocodonAUG

•  Verificaçãodocodondependedebasesaoredor(5’ACCAUGG3’)

Lodisch,7thed

eiF4 complex {

@

5 .• _5? ____ _



formation

D >>----+ @GTP

405


0 }


Met

complex

Attachment to mRNA

AUG

5' to 3' scanning




® ®GOP




eIF2-GTPàeIF2-GDP– associaçãodasubunidademaioresínteseprotéica

eIF2B

Lodisch,7thed

Controledatradução–eIF2

Alberts,5thed

Controledatradução–eIF4E

Fatoresdecrescimentoa5vamproteínasquefosforilamfatoresregulatóriosdeeIF4E:4E-BP4E-BPdesligadeeIF4E:tradução

Controledatradução-IRES

TraduçãodependentedeIRES(internalribosomeentrysite)

Alberts,5thed

Controledatradução-IRES

RNAsviraisassociamcom

eIF4G

Elongaçãodatradução

FatoreseEFHidróliseGTP-eEF1alpha:formaçãodaligaçãopepAdica

Elongaçãodatradução

Translocação:ribossomomove-se3nucleoAdeosposicionandopróximocodonnosí5oAeEF2

Terminaçãodatradução

Alberts,5thed

EstabilidadedeRNAsnocitoplasma

Alberts,5thed

Polirribossomos

Váriosribossomospodemtraduzirapar5rdomesmomRNA(bactériaseeucariotos):polirribossomos(polissomos)

Alberts,5thed

Controledatraduçãoemeucariotos

Incorporaçãodeselenocisteína:dependedesinalnomRNA(estr.secundária)

Alberts,5thed

Growth factor receptor

Cytoplasm

Protein synthesis

••• Nutrients

•

nutrients

Ribosome biogenesis

Pol Ill M acroautophagy transcript ion

FIGURE 8-30 mTOR pathway. mTOR is an active protein kinase when bound by a complex of Rheb and an associated GTP (lower /eft).ln contrast, mTOR is inactive when bound by a complex of Rheb associated with GDP (lower right). When active, the TSCl fTSC2 Rheb-GTPase activating protein (Rheb-GAP) causes hydrolysis of Rheb-bound GTP to GDP, thereby inactivating mTOR. The TSClfTSC2 Rheb-GAP is activated (arrows) by phosphorylation by AMP kinase (AMPK) when cellular energy charge is low and by other cellular stress responses. Signal-transduction pathways activated by cell-surface growth factor receptors lead to phosphorylation of inactivating sites on TSClfTSC2, inhibiting its GAP activity. Consequently, they leave a higher fraction of cellular Rheb in the GTP conformation that activates

Translation of a specific subset of mRNAs that have a string of pyrimidines in their 5' untranslated regions (called TOP mRNAs for tract of oligopyrimidine) is stimulated particularly strongly by mTOR. The T0P mRNAs encode ribosomal pro-teins and translation elongation factors. mTOR also activates the RNA polymerase I transcription factor TIF-lA, stimulating transcription of the large rRNA precursor (see Figure 7-52).

mTOR activates transcription by RNA polymerase III as well, by phosphorylating and thereby activating protein ki-nases that phosphorylate MAFl, a protein inhibitor of RNA polymerase III transcription. MAFl phosphorylation causes it to be exported from the nucleus, relieving repression of RNA polymerase III transcription. When mTOR activity falls , MAFl in the cytoplasm is rapidly dephosphorylated and im-ported into the nucleus where it represses transcription by RNA polymerase III.

In addition, mTOR activates two RNA polymerase II ac-tivators that stimulate transcription of ribosomal protein and

mTOR protein kinase activity. Low nutrient concentration also regulates Rheb GTPase activity, by a mechanism that does not require TSClfTSC2. Active mTOR phosphorylates 4E-BP, causing it to release eiF4E, stimulating translation initiation. It also phosphorylates and activates 56 kinase (S6K), which in turn phosphorylates ribosomal proteins, stimulating translation. Activated mTOR also activates transcription factors for RNA polymerases I, II, and Ill, leading to synthesis and assembly of ribosomes, tRNAs, and translation factors. In the absence of mTOR activity, all of these processes are inhibited. In contrast, activated mTOR inhibits macroautophagy, which is stimulated in cells with inactive mTOR. [Adapted from S. Wullschleger et al., 2006, Cell 124:471.]

translation factor genes. Finally, mTOR stimulates process-ing of the rRNA precursor (Section 8.5). As a consequence of phosphorylation of these several mTOR substrates, the syn-thesis and assembly of ribosomes as well as the synthesis of translation factors and tRNAs are greatly increased. Alterna-tively, when mTOR kinase activity is inhibited, these sub-strates become dephosphorylated, greatly decreasing the rate of protein synthesis and the production of ribosomes, transla-tion factors, and tRNAs, thus halting cell growth.

mTOR activity is regulated by a monomeric small G pro-tein in the Ras protein family called Rheb. Like other small G proteins, Rheb is in its conformation when it is bound to GTP. Rheb·GTP binds the mTOR complex, stimulating mTOR kinase activity, probably by inducing a conformation change in its kinase domain. Rheb is in turn regulated by a heterodimer composed of subunits TSCl and TSC2, named for their involvement in the medical syndrome tuberous scle-rosis complex, as discussed below. In the active conformation,

8.4 Cytoplasmic Mechanisms of Post-transcriptional Control 377

Controledatraduçãoemeucariotos

mTOR

metazoanTargetOfRapamycin

•  Kinase,a5vaquandoligadaaRheb+GTP

•  Respondeàníveisnutricionais

•  Alteradaemdiversos5posdecâncer

Lodisch,7thed

ControledatraduçãoemeucariotosEstruturasecundária5’UTRou3’UTR

Alberts,5thed

Parasabermais

• MolecularBiologyoftheCell–Albertsetal

• GenesVI–Lewin• MolecularCellBiology– Lodisch

• TheCell:Amolecularapproach-Cooper

Tradução I - coltri.bio.br · Tradução: processo pelo qual a sequência de nucleoAdeos de um...

Documents

Transcript of Tradução I - coltri.bio.br · Tradução: processo pelo qual a sequência de nucleoAdeos de um...