lacI

Transcription
TRANSCRIÇÃO
processo 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 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
Nucleotides are added one by one
RNA polymersase catalyses the reaction between the 3’-OH of the growing
strand and the a phosphate of a correctly incoming (base-paired) rNTP
Only one DNA strand, for a specific message,
serves as template during transcription
(or coding strand)
Synthesized RNA is complementary and antiparallel
to one of the two nucleotide strands of DNA
(or anticoding strand)
Template strand is complementary to both coding strand and the transcribed RNA
RNA is transcribed from one DNA strand; gene
organization
Y P R
3’ UAC GCU AGA 5’
ATG CGA TCT
TAC GCT AGA
Gene b
AUG CGA UCU
M
R
S
In most organisms, each gene is transcribed from a single DNA strand
(the template strand), but different genes may be transcribed from
one or other of the two DNA strands
Transcription unit
A
- Promoter
- RNA-coding region
- Terminator
B
A-leader sequence
B- trailler sequence
- Initiation 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
The structure of a bacterial RNA polymerase
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’)
Sigma factor imparts specificity to RNA polymerase attachment to the promoter
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 sequence
is 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 by
the promoter at the begining of each gene,
and though which of the two DNA strands is
to serve as a template
Formação de complexo de transcrição
-Ligação directa
-Via proteínas auxiliares
Sigma factor associates
with the core enzyme to
form the holoenzyme
Closed complex
Unwinding of of dsDNAOpen complex
The sigma factor is
released as the RNA
polymerase moves
from the promoter
Recognition of an E. coli heat shock gene 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 containing
the sigma 70 subunit, but is recognized by the
sigma 32 RNA polymerase that is active during
heat shock.
Sigma factors of E. coli
SIGMA FACTOR PROMOTERS RECOGNIZED
σ70
most genes
σ32
genes induced by heat shock
σ28
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
Interchangeable RNA polymerase subunits as a strategy to control
gene expression in a bacterial virus
Proteins that package
the virus chromosome
SPO I bacterial virus, upon B. subtilis infection uses the bacterial RNA polymerase to transcribe its early genes
Termination of Transcription
Rho-independent
Rho-dependent
Sequence terminator
regions of DNA
An inverted repeat base sequence characterizes
terminator 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 o
número 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 regulation
Positive and negative
Inducible and repressible
CONTROLO NEGATIVO
(Necessidade de um repressor)
INDUZÍVEL (indução)- ex: operão da lactose
OFF
Repressor activo codificado
por um gene
ON
indutor
REPRESSÍVEL (repressão)- ex: operão do trp
Apo-repressor (inactivo) codificado por um gene
(o represssor activo é formado pela interacção
entre o apo-repressor e um co-repressor)
ON
OFF
CONTROLO POSITIVO
(Necessidade de um activador)
OFF
ON
activador
Ex: operão da maltose; operão da lactose
NEGATIVE REGULATION
Inducible and Repressible systems
Vias catabólicas
Ex: operão lac
Vias anabólicas
Ex: operão trp
Induction of
enzyme synthesis
needs the action
of an inducer
Induction of enzyme synthesis
(ex: catabolic pathway of lactose)
(apo-repressor)
Repression of
enzyme synthesis
needs the action
of an apo-repressor
Repression of enzyme synthesis
(ex: anabolic pathway of synthesis of arginine)
Positive regulation
In positive regulation,
the default state of
transcription
is “off”
Positive control
of
enzyme synthesis
An operon is a single transcriptional subunit that includes a series
of structural genes, a promoter and an operator
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
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
P
P
lacI
40
1111
t O
lacZ
lacY
lacA
t
DNA
3063
~ 800
~ 800
bp
RNA
lacI
Polypeptide
Active
Protein
Function
lac
360
38 000
Tetramer
152 000
Repressor
~
1021
125 000
Tetramer
500 000
β-galactosidase
~ 275
~ 275
30 000
30 000
Membrane
Component
30 000
Dimer
60 000
Permease
Transacetylase
Amino acids
Daltons
Daltons
Function
lac operon regulates lactose metabolism
Induction of enzyme synthesis
Lac mutants
lacIlacIs
lacOc
lacZlacP-
Mutations in lacI are constituitive and trans- acting
(merodiploid studies)
lacI+ lacZ- / lacI- lacZ+
In haploid strains,
allows lac
transcription
lacI– , non-fonctional repressor
Cont. lacI-
lacI- is a constituitive 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
The partial diploid lacIS lacZ+ / lacI+ lacZ+ fails to produce βgalactosidase in the presence and absence of lactose, because the
lacIS gene encodes a super-repressor
Transcription
inhibited
lacIS - lactose fails to bind to mutant repressor
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 the
presence and absence
of 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.
A
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.
t
CAP = Proteína activadora do catabolismo
AMPc = Monofosfato cíclico de adenosina
CRP- cAMP receptor protein
Catabolite repression
Diauxic growth and the regulatory elements of the
lac operon
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
In the lac operon, the operator overlaps the promoter
and the 5’ end of the first structural gene
The nucleotide sequence of the regulatory region of the lac
operon, showing regions protected by DNase digestion by the
binding of various proteins
O3