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Why regulation of gene expression is important? •Cellular function is largely dictated by the set of macromolecules inside the cell. •Different macromolecules accumulate to different levels under different growth conditions and in different cell types. •Diseases can be caused by aberrant control of gene expression: too much or too little of a protein; wrong time and wrong place for a protein.
Transcription of DNA into RNA by RNA polymerasean overview 1. Requires DNA template, four ribonucleotide 5’ triphosphates, Mg+2.
Template (non coding) strand
2. De novo synthesis: does not require a primer. Low fidelity compared to DNA polymerase: errors 1/104105 (105 higher than DNA polymerase). 3. Activity highly regulated in vivo: at initiation, elongation and termination. 4. The nucleotide at the 5’ end of an RNA strand retains all three of its phosphate groups; all subsequent nucleotides release pyrophosphate (PPi) when added to the chain and retain only their α phosphate (red). 5. The released PPi is subsequently hydrolyzed by pyrophosphatase to Pi, driving the equilibrium of the overall reaction toward chain elongation. 6. Growth of the transcript always occurs in the 5’to3’ direction.
Nontemplate (coding) strand
E. coli RNA polymerase holoenzyme bound to DNA
ω Subunit Stoichiometry Role in holoenzyme α 2 Binds regulatory sequences/proteins β 1 Forms phosphodiester bonds β’1 σ 1 Promoter recognition ω1 RNAP assembly A single RNA polymerase makes multiple types of RNAs (rRNA, tRNA and mRNA) in prokaryotes.
Typical E.coli promoters recognized by an RNA polymerase holoenzyme containing σ70
Strong promoters: close to consensus sequences and spacing Weak promoters: contain multiple substitutions at the 35 and 10 regions
Dissociation of RNAP and purification of σ by ionexchange chromatography α β σ α β’
[NaCl]
ω
[protein]
Carboxymethyl (CO22) or phospho (PO32) cellulose
Fraction number α α
σ
β ω
β’
The dissociable sigma subunit gives promoter specificity to prokaryotic RNA polymerase (RNAP)
α α
β ω
β’
+
α σ β α β’
σ
ω
Core enzyme
Holoenzyme
No specific promoter binding; tight nonspecific DNA binding (Kd ~5 x 1012 M)
Specific promoter binding; weak nonspecific DNA binding (Kd ~107 M); finds promoter 10,000 times faster.
Transcription initiation by prokaryotic RNA polymerase Holoenzyme α σ α
“sliding and scanning” Promoter 35 10 β β’
Closed complex
α σ β α β’ rNTPs PPi Core enzyme
Open complex; initiation
5’pppA
mRNA
α α
Sigma separates from the core once a few phosphodiester bonds are formed
β β’
σ
Interactions of various sigma factors of E. coli with the same core polymerase to form holoenzymes with different promoter binding specificity Sigma Factor
Promoters Recognized
Promoter Consensus
σ70 σ32 σ28 σ38
Most genes Genes induced by heat shock Genes for motility and chemotaxis Genes for stationary phase and stress response
35 Region 10 Region TTGACAT TATAAT TCTCNCCCTTGAA CCCCATNTA CTAAA CCGATAT ? ?
σ54
24 Region 12 Region Genes for nitrogen metabolism & other functions CTGGNA
TTGCA
Heatshock response:
High temperature induces the production of σ32, which binds to the core polymerase to form a unique holoenzyme for recognition of the promoters of heat shock induced genes.
Rhoindependent prokaryotic transcription termination
The core polymerase pauses after synthesizing a hairpin. If the hairpin is really a terminator, RNA will dissociate from the DNA strand as the AU pairing is unstable. Once the RNA is gone, DNA duplex reforms and the core is driven off, as it has low affinity for dsDNA.
Rhodependent transcription termination
Rhobinding Site (noncontiguous structural features in RNA): Stop signals not recognized by RNAP alone.
Rho: forms RNAdependent hexameric ATPase, translocates along RNA 5’to3’, pulling RNA away when it reaches the transcription bubble.
Platt, Ann. Rev. Biochem. 55: 339 (1986)
termination
Three types of RNA polymerase in eukaryotic nuclei Type
Location
RNA synthesized
Effect of αamanitin
I II III
Nucleolus Nucleoplasm Nucleoplasm
PrerRNA for 18, 5.8 and 28S rRNAs Insensitive PremRNA, some snRNAs Sensitive to 1 µg/ml PretRNAs, 5S rRNA, some snRNAs Sensitive to 10 µg/ml
• αamanitin from Amanita Phalloides binds tightly to RNA Pol II and blocks transcriptional elongation. • RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S prerRNA is present in tandem array. • RNA Pol II transcribe ~25,000 genes; • RNA Pol III transcribe 3050 genes at variable copy numbers.
(Also Organelle RNAPs in Mitochondria and Chloroplasts. Encoded by organelle genomes. Similar to bacterial RNAPs.)
Comparison of 3D structures of bacterial and eukaryotic RNA polymerases
Transcription start sites can be mapped by S1 protection and primer extension Obtained in vitro or in vivo
Red dot: 32P end label
•Mapping the start site for synthesis of a particular mRNA often helps identify the DNA regulatory sequences that control its transcription, because some of the regulatory elements are located near the start site. •The position of the start site can be mapped from the length of the labeled probe segment protected from S1 digestion (S1 protection) or the resulting extension product (primer extension) on polyacrylamideurea gel (same as DNA sequencing gel).
Core Promoter Elements
In vitro stepwise assembly of the RNA Pol II preinitiation complex (PIC) at core promoter for basal transcription
Basal (‘General’) Transcription Factors for RNA Polymerase II
Total: 4344 polypeptides and over 2 million daltons.
“Gel shift”: electrophoretic mobility shift assay (EMSA) for studying DNAprotein interactions
* ProteinDNA complex
* Free DNA probe 1. 2. 3.
Prepare labeled DNA probe Bind protein Native gel electrophoresis
Advantage: sensitive
ON: protein mixture loaded onto an ionexchange column. Fraction 122: fractions eluted from the column with increasing salt concentrations.
Disadvantage: requires stable complex; little information about where protein is binding on DNA
Stepwise assembly of preinitiation complex (PIC) as revealed by gel shift assay
Eukaryotic transcription cycle Only the unphosphorylated RNA Pol II enters PIC.
The TFIIH complex has both helicase and kinase activities that can unwind DNA and phosphorylate the CTD tail of RNA Pol II.
Release of TFIIE and then IIH during the synthesis of the initial 6070nt.
CTD
FCP1
Recycling
Pol II
Pol II
CTD
PTEFb
TFIIH
CTD
5
CDK9
5 5 5
+1
PIC assembly
CycT1
CTD
Pol II
Pol II
2 5 2 5 2 2 5 5
Pol II 5’ cap
Promoter clearance & pausing for capping
2 5 2 5 2 2 5 5
Pol II
2 5 2 5 2 2 5 5
RNA
Release from pausing
Productive elongation
2 CTD heptapeptide repeats: 2752 x (YS PTS5PS)
Cisacting control elements
(a) Genes of multicellular organisms contain both promoterproximal elements and enhancers (collectively referred to as cicacting control elements) as well as a TATA box or other core promoter element(s). (b) Enhancers function in a distance, position and orientationindependent manner. Long distance interactions are achieved by forming looped DNA. (c) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site. (Also note: many yeast genes do not contain introns). (d) In multicellular organisms, one standard promoterproximal element is a GCbox (GGGCGGGC) recognized by the “constitutive” transcriptional activator Sp1.
DNA affinity chromatography for purification of Sp1
DNase I footprinting: a common technique for identifying proteinbinding sites in DNA. 1. A DNA fragment is labeled at one end with 32 P (red dot). 2. Portions of the sample then are digested with DNase I in the presence and absence of a protein that binds to a specific sequence in the fragment. 3. A low concentration of DNase I is used so that on average each DNA molecule is cleaved just once (vertical arrows).
4. The two samples of DNA then are separated from protein, denatured to separate the strands, and electrophoresed. The resulting gel is analyzed by autoradiography, which detects only labeled strands and reveals fragments extending from the labeled end to the site of cleavage by DNase I.
Analyses of affinitypurified Sp1 protein DNase I footprint on SV40 promoter
SDSPAGE/ silverstain
Sp1
Lane 2 contains total cell proteins prior to affinity purification; Lanes 3&4 contain purified Sp1 protein washed off the affinity column.
NaCl
In vitro transcription assay to measure Sp1 activity •The adenovirus DNA template used here does not contain any Sp1binding sites (GCbox) and is therefore used as a negative control. •In vitro transcription reactions contain template DNA, labeled ribonucleoside triphosphates, and purified general transcription factors and RNA Pol II. Purified Sp1 is added to the reactions indicated with “+”.
In vivo assay for transcription factor activity • Host cells should lack the
gene encoding protein X and the reporter protein.
• The production of reportergene RNA transcripts or the activity of the encoded protein can be assayed.
cotransfection
Reportergene products
• If reportergene transcription is greater in the presence of the X encoding plasmid, then X is an activator; if transcription is less, then X is a repressor.
The Production of DNA Microarrays and Their Use in Monitoring Global Gene Transcription
Regulation of prokaryotic transcription 1.
Singlecelled organisms with short doubling times must respond extremely rapidly to their environment.
2.
Halflife of most mRNAs is short (on the order of a few minutes).
5.
Coupled transcription and translation occur in a single cellular compartment.
Therefore, transcription initiation is usually the major control point. Most prokaryotic genes are regulated in units called operons (Jacob and Monod, 1960) Operon: a coordinated unit of gene expression consisting of one or more related genes and the operator and promoter sequences that regulate their transcription. The mRNAs thus produced are “polycistronic’—multiple genes on a single transcript.
The trp operon: two kinds of negative regulation
(low trp levels)
(high trp levels)
Tryptophan + trp repressor dimer Trprepressor complex activated for DNA binding Binds Operator; blocks RNAP binding & represses transcription; Tryptophan a corepressor
Translation of part of the leader mRNA to produce the leader peptide
The attenuator is a Rhoindependent transcription terminator! What is the attenuator?
Presence of Trp codons within the leader peptide is highly significant!
Attenuation is mediated by the tight coupling of transcription and translation •The ribosome translating the trp leader mRNA follows closely behind the RNA polymerase that is transcribing the DNA template. •Alternative conformation adopted by the leader mRNA.
completed
UUU 3’ and blocking sequence 2 Incomplete leader peptide
•The stalled ribosome is waiting for tryptophanyl tRNA. •The 2:3 pair is not an attenuator and is more stable than the 3:4 pair.
Twostep decoding process for translating nucleic acid sequences in mRNA into amino acid sequences in proteins
The PhoR/PhoB twocomponent regulatory system in E. coli
In response to low phosphate concentrations in the environment and periplasmic space, a phosphate ion dissociates from the periplasmic domain of the sensor protein PhoR. This causes a conformational change that activates a protein kinase transmitter domain in the cytosolic region of PhoR. The activated transmitter domain transfers an ATP γphosphate to a histidine in the transmitter domain. This phosphate is then transferred to an aspartic acid in the response regulator PhoB. Phosphorylated PhoB then activate transcription from genes encoding proteins that help the cell to respond to low phosphate, including phoA, phoS, phoE, and ugpB.
Activation of σ 54containing RNA polymerase at glnA promoter by NtrC
(kinase)
•The glnA gene encodes glutamine synthetase, which synthesizes glutamine from glutamic acid and ammonia. • The σ54containing RNA polymerase binds to the glnA promoter, forming a closed complex, before being activated. • In response to low levels of glutamine, a protein kinase called NtrB phosphorylates dimeric NtrC, which then binds to two sequence elements (called enhancer) located at –108 and 140. • The bound phosphorylated NtrC dimers interact with the bound σ54polymerase, causing the intervening DNA to form a loop. • The ATPase activity of NtrC then stimulates the polymerase to unwind the template strands at the start site, forming an open complex. Transcription of the glnA gene can then begin.
Transcription of DNA into RNA by RNA polymerasean overview 1. Requires DNA template, four ribonucleotide 5’ triphosphates, Mg+2.
Template (non coding) strand
2. De novo synthesis: does not require a primer. Low fidelity compared to DNA polymerase: errors 1/104105 (105 higher than DNA polymerase). 3. Activity highly regulated in vivo: at initiation, elongation and termination. 4. The nucleotide at the 5’ end of an RNA strand retains all three of its phosphate groups; all subsequent nucleotides release pyrophosphate (PPi) when added to the chain and retain only their α phosphate (red). 5. The released PPi is subsequently hydrolyzed by pyrophosphatase to Pi, driving the equilibrium of the overall reaction toward chain elongation. 6. Growth of the transcript always occurs in the 5’to3’ direction.
Nontemplate (coding) strand
E. coli RNA polymerase holoenzyme bound to DNA
ω Subunit Stoichiometry Role in holoenzyme α 2 Binds regulatory sequences/proteins β 1 Forms phosphodiester bonds β’1 σ 1 Promoter recognition ω1 RNAP assembly A single RNA polymerase makes multiple types of RNAs (rRNA, tRNA and mRNA) in prokaryotes.
Typical E.coli promoters recognized by an RNA polymerase holoenzyme containing σ70
Strong promoters: close to consensus sequences and spacing Weak promoters: contain multiple substitutions at the 35 and 10 regions
Dissociation of RNAP and purification of σ by ionexchange chromatography α β σ α β’
[NaCl]
ω
[protein]
Carboxymethyl (CO22) or phospho (PO32) cellulose
Fraction number α α
σ
β ω
β’
The dissociable sigma subunit gives promoter specificity to prokaryotic RNA polymerase (RNAP)
α α
β ω
β’
+
α σ β α β’
σ
ω
Core enzyme
Holoenzyme
No specific promoter binding; tight nonspecific DNA binding (Kd ~5 x 1012 M)
Specific promoter binding; weak nonspecific DNA binding (Kd ~107 M); finds promoter 10,000 times faster.
Transcription initiation by prokaryotic RNA polymerase Holoenzyme α σ α
“sliding and scanning” Promoter 35 10 β β’
Closed complex
α σ β α β’ rNTPs PPi Core enzyme
Open complex; initiation
5’pppA
mRNA
α α
Sigma separates from the core once a few phosphodiester bonds are formed
β β’
σ
Interactions of various sigma factors of E. coli with the same core polymerase to form holoenzymes with different promoter binding specificity Sigma Factor
Promoters Recognized
Promoter Consensus
σ70 σ32 σ28 σ38
Most genes Genes induced by heat shock Genes for motility and chemotaxis Genes for stationary phase and stress response
35 Region 10 Region TTGACAT TATAAT TCTCNCCCTTGAA CCCCATNTA CTAAA CCGATAT ? ?
σ54
24 Region 12 Region Genes for nitrogen metabolism & other functions CTGGNA
TTGCA
Heatshock response:
High temperature induces the production of σ32, which binds to the core polymerase to form a unique holoenzyme for recognition of the promoters of heat shock induced genes.
Rhoindependent prokaryotic transcription termination
The core polymerase pauses after synthesizing a hairpin. If the hairpin is really a terminator, RNA will dissociate from the DNA strand as the AU pairing is unstable. Once the RNA is gone, DNA duplex reforms and the core is driven off, as it has low affinity for dsDNA.
Rhodependent transcription termination
Rhobinding Site (noncontiguous structural features in RNA): Stop signals not recognized by RNAP alone.
Rho: forms RNAdependent hexameric ATPase, translocates along RNA 5’to3’, pulling RNA away when it reaches the transcription bubble.
Platt, Ann. Rev. Biochem. 55: 339 (1986)
termination
Three types of RNA polymerase in eukaryotic nuclei Type
Location
RNA synthesized
Effect of αamanitin
I II III
Nucleolus Nucleoplasm Nucleoplasm
PrerRNA for 18, 5.8 and 28S rRNAs Insensitive PremRNA, some snRNAs Sensitive to 1 µg/ml PretRNAs, 5S rRNA, some snRNAs Sensitive to 10 µg/ml
• αamanitin from Amanita Phalloides binds tightly to RNA Pol II and blocks transcriptional elongation. • RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S prerRNA is present in tandem array. • RNA Pol II transcribe ~25,000 genes; • RNA Pol III transcribe 3050 genes at variable copy numbers.
(Also Organelle RNAPs in Mitochondria and Chloroplasts. Encoded by organelle genomes. Similar to bacterial RNAPs.)
Comparison of 3D structures of bacterial and eukaryotic RNA polymerases
Transcription start sites can be mapped by S1 protection and primer extension Obtained in vitro or in vivo
Red dot: 32P end label
•Mapping the start site for synthesis of a particular mRNA often helps identify the DNA regulatory sequences that control its transcription, because some of the regulatory elements are located near the start site. •The position of the start site can be mapped from the length of the labeled probe segment protected from S1 digestion (S1 protection) or the resulting extension product (primer extension) on polyacrylamideurea gel (same as DNA sequencing gel).
Core Promoter Elements
In vitro stepwise assembly of the RNA Pol II preinitiation complex (PIC) at core promoter for basal transcription
Basal (‘General’) Transcription Factors for RNA Polymerase II
Total: 4344 polypeptides and over 2 million daltons.
“Gel shift”: electrophoretic mobility shift assay (EMSA) for studying DNAprotein interactions
* ProteinDNA complex
* Free DNA probe 1. 2. 3.
Prepare labeled DNA probe Bind protein Native gel electrophoresis
Advantage: sensitive
ON: protein mixture loaded onto an ionexchange column. Fraction 122: fractions eluted from the column with increasing salt concentrations.
Disadvantage: requires stable complex; little information about where protein is binding on DNA
Stepwise assembly of preinitiation complex (PIC) as revealed by gel shift assay
Eukaryotic transcription cycle Only the unphosphorylated RNA Pol II enters PIC.
The TFIIH complex has both helicase and kinase activities that can unwind DNA and phosphorylate the CTD tail of RNA Pol II.
Release of TFIIE and then IIH during the synthesis of the initial 6070nt.
CTD
FCP1
Recycling
Pol II
Pol II
CTD
PTEFb
TFIIH
CTD
5
CDK9
5 5 5
+1
PIC assembly
CycT1
CTD
Pol II
Pol II
2 5 2 5 2 2 5 5
Pol II 5’ cap
Promoter clearance & pausing for capping
2 5 2 5 2 2 5 5
Pol II
2 5 2 5 2 2 5 5
RNA
Release from pausing
Productive elongation
2 CTD heptapeptide repeats: 2752 x (YS PTS5PS)
Cisacting control elements
(a) Genes of multicellular organisms contain both promoterproximal elements and enhancers (collectively referred to as cicacting control elements) as well as a TATA box or other core promoter element(s). (b) Enhancers function in a distance, position and orientationindependent manner. Long distance interactions are achieved by forming looped DNA. (c) Most yeast genes contain only one regulatory region, called an upstream activating sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site. (Also note: many yeast genes do not contain introns). (d) In multicellular organisms, one standard promoterproximal element is a GCbox (GGGCGGGC) recognized by the “constitutive” transcriptional activator Sp1.
DNA affinity chromatography for purification of Sp1
DNase I footprinting: a common technique for identifying proteinbinding sites in DNA. 1. A DNA fragment is labeled at one end with 32 P (red dot). 2. Portions of the sample then are digested with DNase I in the presence and absence of a protein that binds to a specific sequence in the fragment. 3. A low concentration of DNase I is used so that on average each DNA molecule is cleaved just once (vertical arrows).
4. The two samples of DNA then are separated from protein, denatured to separate the strands, and electrophoresed. The resulting gel is analyzed by autoradiography, which detects only labeled strands and reveals fragments extending from the labeled end to the site of cleavage by DNase I.
Analyses of affinitypurified Sp1 protein DNase I footprint on SV40 promoter
SDSPAGE/ silverstain
Sp1
Lane 2 contains total cell proteins prior to affinity purification; Lanes 3&4 contain purified Sp1 protein washed off the affinity column.
NaCl
In vitro transcription assay to measure Sp1 activity •The adenovirus DNA template used here does not contain any Sp1binding sites (GCbox) and is therefore used as a negative control. •In vitro transcription reactions contain template DNA, labeled ribonucleoside triphosphates, and purified general transcription factors and RNA Pol II. Purified Sp1 is added to the reactions indicated with “+”.
In vivo assay for transcription factor activity • Host cells should lack the
gene encoding protein X and the reporter protein.
• The production of reportergene RNA transcripts or the activity of the encoded protein can be assayed.
cotransfection
Reportergene products
• If reportergene transcription is greater in the presence of the X encoding plasmid, then X is an activator; if transcription is less, then X is a repressor.
The Production of DNA Microarrays and Their Use in Monitoring Global Gene Transcription
Regulation of prokaryotic transcription 1.
Singlecelled organisms with short doubling times must respond extremely rapidly to their environment.
2.
Halflife of most mRNAs is short (on the order of a few minutes).
5.
Coupled transcription and translation occur in a single cellular compartment.
Therefore, transcription initiation is usually the major control point. Most prokaryotic genes are regulated in units called operons (Jacob and Monod, 1960) Operon: a coordinated unit of gene expression consisting of one or more related genes and the operator and promoter sequences that regulate their transcription. The mRNAs thus produced are “polycistronic’—multiple genes on a single transcript.
The trp operon: two kinds of negative regulation
(low trp levels)
(high trp levels)
Tryptophan + trp repressor dimer Trprepressor complex activated for DNA binding Binds Operator; blocks RNAP binding & represses transcription; Tryptophan a corepressor
Translation of part of the leader mRNA to produce the leader peptide
The attenuator is a Rhoindependent transcription terminator! What is the attenuator?
Presence of Trp codons within the leader peptide is highly significant!
Attenuation is mediated by the tight coupling of transcription and translation •The ribosome translating the trp leader mRNA follows closely behind the RNA polymerase that is transcribing the DNA template. •Alternative conformation adopted by the leader mRNA.
completed
UUU 3’ and blocking sequence 2 Incomplete leader peptide
•The stalled ribosome is waiting for tryptophanyl tRNA. •The 2:3 pair is not an attenuator and is more stable than the 3:4 pair.
Twostep decoding process for translating nucleic acid sequences in mRNA into amino acid sequences in proteins
The PhoR/PhoB twocomponent regulatory system in E. coli
In response to low phosphate concentrations in the environment and periplasmic space, a phosphate ion dissociates from the periplasmic domain of the sensor protein PhoR. This causes a conformational change that activates a protein kinase transmitter domain in the cytosolic region of PhoR. The activated transmitter domain transfers an ATP γphosphate to a histidine in the transmitter domain. This phosphate is then transferred to an aspartic acid in the response regulator PhoB. Phosphorylated PhoB then activate transcription from genes encoding proteins that help the cell to respond to low phosphate, including phoA, phoS, phoE, and ugpB.
Activation of σ 54containing RNA polymerase at glnA promoter by NtrC
(kinase)
•The glnA gene encodes glutamine synthetase, which synthesizes glutamine from glutamic acid and ammonia. • The σ54containing RNA polymerase binds to the glnA promoter, forming a closed complex, before being activated. • In response to low levels of glutamine, a protein kinase called NtrB phosphorylates dimeric NtrC, which then binds to two sequence elements (called enhancer) located at –108 and 140. • The bound phosphorylated NtrC dimers interact with the bound σ54polymerase, causing the intervening DNA to form a loop. • The ATPase activity of NtrC then stimulates the polymerase to unwind the template strands at the start site, forming an open complex. Transcription of the glnA gene can then begin.
