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
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