Transcription

TRANSCRIÇÃOprocesso de síntese de RNA a partir de uma molécula de DNA molde, usando as regras de complementaridade

RNA synthesis

Initiaton of RNA synthesis does not require a primer

New nucleotides are added to the 3’ end of the DNA molecule

DNA unwinds at the front of the transcription bubble and then rewinds

Ribonucleoside triphosphate are substrates used inRNA synthesis

Polymerization of ribonucleotides by RNA polymeraseduring transcription

Incoming ribonucleoside triphosphate

The energy stored in their P-P bonds provides thedriving force for the polymerization reaction

RNA polymersase catalyses the reaction between the 3’-OH of the growingstrand and the a phosphate of a correctly incoming (base-paired) rNTP

Nucleotides are added one by one

Synthesized RNA is complementary and antiparallelto one of the two nucleotide strands of DNA

(or anticoding strand)

Template strand is complementary to both coding strand and the transcribed RNA

Only one DNA strand, for a specific message, serves as template during transcription

(or coding strand)

RNA is transcribed from one DNA strand; gene organization

In most organisms, each gene is transcribed from a single DNA strand(the template strand), but different genes may be transcribed fromone or other of the two DNA strands

ATG CGA TCT

AUG CGA UCU

TAC GCT AGA

M R S

5’3’ UAC GCU AGAY P R

Gene b

Transcription unit

- Promoter- RNA-coding region- Terminator

BA

A-leader sequenceB- trailler sequence

- Initiation codon

DNA is transcribed by the enzymeRNA polymerase

Polarity of synthesized RNA strands (5’-3’) is opposite to their template DNA strands

A short region of DNA/RNA helix (approx. 9 nts in lenght) is formed only transiently.A window of this DNA/RNA helix moves along the DNA with the polymerase

RNA polymerase unwinds the DNA helix at its active site and moves stepwise along

The structure of a bacterial RNA polymerase

Sigma factor imparts specificity to RNA polymerase attachment to the promoter

In bacterial RNA polymerse, the core enzyme consists of four subunits: two copies of alpha (α), a single copy of beta (β), and a single copy of

beta prime (β’)

In bacterial promoters, consensus sequences are found upstream of the start site, approximately at

positions-10 and -35

+1 first base transcribed

Bacterial promoters

In most prokaryotic promoters, the actual – 10 sequenceis not TATAAT

Molecular model: RNA polymerase (in yellow) binding DNA

Directions of transcription along a short portion of a bacterial chromosome

Direction of transcription is determined bythe promoter at the begining of each gene,and though whichwhich ofof thethe twotwo DNA DNA strandsstrands isisto serve as a to serve as a templatetemplate

Formação de complexo de transcrição-Ligação directa-Via proteínas auxiliares

Sigma factor associateswith the core enzyme toform the holoenzyme

Closed complex

Unwinding of of dsDNA-Open complex

The sigma factor isreleased as the RNA polymerase moves from the promoter

Recognition of an E. coli heat shock gene by thesigma 32 subunit

The sequence of the heat-shock promoter isdifferent from that of the normal E. coli promoter

The heat-shock promoter is not recognized by the normal E. coli RNA polymerase containingthe sigma 70 subunit, but is recognized by thesigma 32 RNA polymerase that is active duringheat shock.

SIGMA FACTOR PROMOTERS RECOGNIZED

σ70 most genesσ32 genes induced by heat shockσ28 genes for stationary phase and stress responseσ28 genes involved in motility and chemotaxisσ54 genes for nitrogen metabolism

The sigma factor designations refer to their approximate molecular weights, in kDa

Sigma factors of E. coli

Interchangeable RNA polymerase subunits as a strategy to controlgene expression in a bacterial virus

SPO I bacterial virus, upon B. subtilis infection uses the bacterial RNA polymerase to transcribe its early genes

Proteins that packagethe virus chromosome

Termination of Transcription

Rho-independentRho-dependent

Sequence terminatorregions of DNA

An inverted repeat base sequence characterizesterminator regions of DNA.

Stem-loop structures can occur as the RNA formsbecause of complementary sequences.

The 3’ poly-U tail indicates a rho-independentterminator

Formação de RNA-RNA reduz onúmero de contactos entre molde e transcrito e promove a dissociaçãoda RNA polimerase

Rho is a helicase that follows RNA polymerase along the transcript. When the polymerasestalls at a hairpin, Rho catches up and breaks the RNA/DNA bp, releasing the transcript.

Rho-dependent termination of transcription

Transcription regulation

Positive and negativeInducible and repressible

CONTROLO NEGATIVO(Necessidade de um repressor)

INDUZÍVEL (indução)- ex: operão da lactoseOFF ON

indutor

REPRESSÍVEL (repressão)- ex: operão do trpON OFF

CONTROLO POSITIVO (Necessidade de um activador)

OFF ON

activador

Ex: operão da maltose; operão da lactose

Repressor activo codificado por um gene

Apo-repressor (inactivo) codificado por um gene(o represssor activo é formado pela interacção entre o apo-repressor e um co-repressor)

NEGATIVE REGULATIONInducible and Repressible systems

Vias catabólicasEx: operão lac

Vias anabólicasEx: operão trp

Induction ofenzyme synthesisneeds the actionof an inducer

Induction of enzyme synthesis(ex: catabolic pathway of lactose)

Repression ofenzyme synthesisneeds the action of an apo-repressor

(apo-repressor)

Repression of enzyme synthesis(ex: anabolic pathway of synthesis of arginine)

Positive regulation

In positive regulation,the default state oftranscriptionis “off”

Positive controlofenzyme synthesis

An operon is a single transcriptional subunit that includes a seriesof structural genes, a promoter and an operator

In some operons, product molecules may bind to the regulator protein either to activate it or turn it off

Separate regulator gene with its ownpromoter

lac operon

an example of a negative inducible andsimultaneously positive mechanisms of

transcription regulation

Lactose, a major carbohydrate found in milk, consistsof 2 six-carbon sugars linked together

β-1,4 bond

β-1,6 bond

Map of the lac operon

The lacA gene is not essential for lactose utilization

The lac operon occupies 6000 bp of DNA

Polypeptide

Active Protein

Function

Amino acidsDaltons

Daltons

Functionβ-galactosidaseRepressor TransacetylasePermease

36038 000

MembraneComponent30 000

Dimer60 000

~ 27530 000

~ 27530 000

Tetramer500 000

Tetramer152 000

1021125 000

~

DNA

RNA

P lacI t O lacZ lacY lacA t

P

bp

lacI lac

1111 3063 ~ 800 ~ 80040

lac operon regulates lactose metabolism

Induction of enzyme synthesis

Lac mutants

lacI-lacIslacOc

lacZ-

lacP-

Mutations in lacI are constituitive and trans- acting(merodiploid studies)

lacI– , non-fonctional repressor

In haploid strains,allows lactranscription

lacI+ lacZ- / lacI- lacZ+

Cont. lacI-

lacI- is a constituitive mutant:in haploid strains expression of lac occurs either in thepresence and absenceof lactose

The partial diploid lacI+ lacZ- / lacI- lacZ+

produces β-galactosidade only in the presenceof lactose because the lacI gene is trans-dominant

The partial diploid lacIS lacZ+ / lacI+ lacZ+ fails to produce β-galactosidase in the presence and absence of lactose, because the

lacIS gene encodes a super-repressor

lacIS - lactose fails to bind to mutant repressor

Transcriptioninhibited

lacIS

Mutations in lacO are constituitive and cis acting

lacOc - non-fonctional operator:repressor fails to bind to operator

Cont. lacOc

lacOc -is a constituitive mutant: in haploid strains expression of lac occurs either in thepresence and absenceof lactose

Cont. lacOc

Cont. lacOc

lacO gene is cis acting

Positive control

Controlo positivo do operão lac de Escherichia coli

Os genes do operão lac não se exprimem se o meio de crescimento contiver glucose.

A glucose exerce repressão catabólica no operão lac.

Uma vez a glucose esgotada há indução do operão lac.

t

AO operão lac não tem um promotor forte.

Para ser reconhecido pela RNA polimerase é necessário que um factor auxiliar esteja ligado ao DNA num local adjacente.

O factor auxiliar da transcrição é o complexo CAP-AMP cíclico.

CAP = Proteína activadora do catabolismo

AMPc = Monofosfato cíclico de adenosina

CRP- cAMP receptor protein

Catabolite repression

Diauxic growth and the regulatory elements of thelac operon

The catabolite activator protein (CAP) binds to thepromoter of the lac operon and stimulates transcription

Binding of the cAMP-CAP complex to DNA produces a sharp bendin DNA that activates transcription

In the lac operon, the operator overlaps the promoterand the 5’ end of the first structural gene

The nucleotide sequence of the regulatory region of the lacoperon, showing regions protected by DNase digestion by the

binding of various proteins

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