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