Transcription

TRANSCRIÇÃO

. processo de síntese de RNA

. a partir de uma molécula de DNA molde,

. usando as regras de complementaridade

. a molécula de RNA sintetizada não segue a regra

de Chargaffde Chargaff

. é um processo que envolve diferentes proteinas

. RNA em todos os organismos vivos é sintetizado

por uma RNA polimerase dependente do DNA (RNAP)

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 in RNA synthesis

Polymerization of ribonucleotides by RNA polymerase during transcription

Incoming ribonucleoside triphosphate

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

RNA polymersase catalyses the reaction between the 3’-OH of the growing

strand and the a phosphate of a correctly incoming (base-paired) rNTP

Nucleotides are added one by one

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

(or coding strand)

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

Each gene is transcribed from

a single DNA strandbut different genes may be transcribed from

one or other of the two DNA strands

5’3’ UAC GCU AGAY P R

ATG CGA TCT

AUG CGA UCU

TAC GCT AGA

M R S

5’3’ UAC GCU AGA

Gene b

Overview of transcription

Transcription unit

- Promoter- RNA-coding region- Terminator

BA

A-leader sequence or 5’-UTR (untranslated)

B- trailler sequence or 3’-UTR

- Initiation codon

- Termination codon

DNA is transcribed by the enzyme RNA polymerase

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

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

In bacterial RNA polymerse, the core enzyme consists of five subunits: two copies of alpha (αααα), a single copy of beta (ββββ), a single copy of beta prime (ββββ’) and a single copy

of omega (ω)ω)ω)ω)

Sigma factor imparts specificity to RNA polymerase attachment to the promoter

ωωωω ωωωω

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

(ex. of consensus sequence recognized by sigma 70 localized approximately at positions -10 and -

35)

+1 first base transcribed

Bacterial promoters

In most prokaryotic promoters, the actual – 10 sequence is not TATAAT

Direction of transcription

is determined by

the promoter at the begining of each gene,

and though

which of the two DNA strands is to serve as a templatewhich of the two DNA strands is to serve as a template

Many RNAs can be simultaneously transcribed from a gene

Sigma factor associates

with the core enzyme to

form the holoenzyme

Closed complex

Unwinding of of dsDNA-

Open complex

The sigma factor is

released as the RNA

polymerase moves

from the promoter

Recognition of an E. coli heat shock genes by the

sigma 32 subunit

The sequence of the heat-shock promoter is different 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 genes

σ32 genes induced by heat shock

Sigma factors of E. coli

σ38 genes for stationary phase and stress response

σ28 genes involved in motility and chemotaxis

σ54 genes for nitrogen metabolism

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

The holoenzymes with the minor σ-factors transcribe discrete

sets of operons and regulons

in response to specific physiological requirements

Interchangeable RNA polymerase subunits as a strategy to control gene 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-independent

Rho-dependent

Sequence terminator regions of DNA

An inverted repeat base sequence characterizesterminator regions of DNA.

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

The 3’ poly-U tail indicates a rho-independent terminator

Formação de RNA-RNA reduz onúmero de contactos entre molde

e transcrito e promove a dissociação

da RNA polimerase

Rho-dependent termination of transcription

Rho is a helicase that follows RNA polymerase along the transcript. When the polymerase

stalls at a hairpin, Rho catches up and breaks the RNA/DNA bp, releasing the transcript.

Transcription unit

- Promoter- RNA-coding region- Terminator

BA

A-leader sequence or 5’-UTR (untranslated)

B- trailler sequence or 3’-UTR

- Initiation codon

- Termination codon

Transcription regulation

Positive and negative

Inducible and repressible

CONTROLO NEGATIVO(Necessidade de um repressor)

INDUZÍVEL (indução)- ex: operão da lactose

OFF ON

indutor

REPRESSÍVEL (repressão)- ex: operão do trp

ON OFF

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)

CONTROLO POSITIVO (Necessidade de um activador)

OFF ON

activador

Ex: operão da maltose; operão da lactose

entre o apo-repressor e um co-repressor)

NEGATIVE REGULATIONInducible and Repressible systems

Cis-acting elements

Ex. operators

Catabolic pathwaysEx. lac operon

Anabolic pathways

Ex. trp operon

NEGATIVE REGULATION

Inducible system

(ex: catabolic pathway of lactose)

Induction of enzyme synthesisneeds the action of an inducer

Lactose is the inducer

NEGATIVE REGULATION

Repressible systems

(ex: anabolic pathway of synthesis of arginine)

Repression of enzyme synthesisneeds the action of co-repressor

(apo-repressor)

Arginine is the co-repressor

Positive controlIn positive regulation,

the default state of

transcription is “off”

The regulator protein (activator)

promotes the binding of RNAP

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

(malE) (malF) (malG)

An operon is a single transcriptional subunit that includes a series of structural genes, which are under the same transcriptional control

Separate regulator gene with its own promoter

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

lac operon

An example of a

negative inducible

and simultaneously

positive

mechanisms of transcription regulation

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

ββββ-1,4 bond

ββββ-1,6 bond

Map of the lac operon

Absence of lactose

The lacA gene is not essentialfor lactose utilization

Presence of lactose

lac operon regulates lactose metabolism

Induction of enzyme synthesis

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

lacZ

The lac operon occupies 6000 bp of DNA

~~

DNA

RNA

P lacI t O lacZ lacY lacA t

P

bp

lacI lac

1111 3063 ~ 800 ~ 80040

Polypeptide

Active Protein

Function

Amino acidsDaltons

Daltons

Functionββββ-galactosidaseRepressor TransacetylasePermease

36038 000

Membrane Component30 000

Dimer60 000

~ 275

30 000

~ 275

30 000

Tetramer500 000

Tetramer152 000

1021125 000

Lac mutants

lacI-

lacIs

lacOc

lacZ-

lacP-

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

lacI+ lacZ- / lacI- lacZ+

lacI– , non-functional repressor

In haploid strains, allows lac transcription,

even in absence of lactose

Cont. lacI-

lacI- is a constitutive mutant:

in haploid strains expression

of lac occurs either in the

presence and absence

of lactose

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

produces β-galactosidade only in the presence

of lactose because the lacI gene is trans-dominant

lacIS encodes a super-repressor

The partial diploid

fails to produce β-galactosidase

in the presence and absence of lactose

lacIS lacZ+ / lacI+ lacZ+

lacIS - lactose fails to bind to mutant repressor

Transcription

inhibited

Mutations in lacO are constitutive 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 the

presence 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.

O operão lac não tem um promotor forte.

t

A

O 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

Diauxic growth if two energy sources are present in the medium at the same time.

The enzyme(s) needed for utilization of one of the energy sources is subject to

catabolite repression.

Catabolite repression

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

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

The nucleotide sequence of the regulatory region of the lac operon, showing regions protected by DNase digestion by the binding of various

proteins

O3