transcriptional and post transcriptional regulation of gene expression
TRANSCRIPT
Transcriptional and post transcriptional regulation of gene expression
Presented by : Kirti Ph.D. (MBB)
What is gene expression ?
• It is the process by which information from a gene is used in the synthesis of a functional gene product.
• These products are often proteins, but in non coding genes such as rRNA genes or tRNA genes, the product is functional RNA.
• Prokaryotic organisms regulate gene expression in response to their environment.
• Eukaryotic cells regulate gene expression to maintain homeostasis in the organism.
Classification of genes with respect to their expression
Constitutively expressed genes ( housekeeping genes ) :
• Are expressed at fixed rate, irrespective of cell condition.• The genes that are involved in vital biochemical
processes such as respiration.• Their structure is simpler. Regulated gene expression (Controllable genes) :
• Are expressed only as needed. Their amount may increase or decrease with respect to their basal level in different condition.
• Their structure is relatively complicated with some response elements.
An overview of Transcription
.
How is gene expression controlled?
• Gene activity is controlled first and foremost at the level of transcription.
• Much of this control is achieved through the interplay between proteins that bind to specific DNA sequences and their DNA binding sites.
• Gene-regulation mechanisms in prokaryotes particularly in E. coli have been extensively investigated.
Transcription is more selective
During Replication the entire chromosome is usually copied but transcription is more selective
Only particular genes or group of genes are transcribed at any one time and some portions of DNA genome are never transcribed
Thus cell restricts the expression of genetic information to the formation of gene products needed at any particular moment
Modern microarray analysis of transcription pattern has revealed that much of genome of humans and other mammals is transcribed into RNA
involved in regulation of gene expression
3 Phases of Transcription : Initiation Elongation TerminationInitiation phases of DNA binding initiation of RNA synthesis
Structure and nomenclature of a typical gene
Most genes are made up of 3 regions
Structural gene• Centre sequence that is copied in the form of RNA• Concerned with the synthesis of polypeptide chain or a
number of polypeptide chains • In Eukaryotes, these occur in spilt-form, segmented into
introns and exons• In Prokaryotes, these are continuous
Structural gene
Promoter region
Terminator region
Promoter sequences in DNA Regulatory region of DNA usually located to the 5 ’ end or upstream from
structural gene well characterised in prokaryotes such as E.coli binding of transcription factors comprise two highly conserved sequences 1. Pribnow box or the TATA box (six nucleotides consensus sequence
TATAAT) 10 bp upstream from the transcription initiation site (-10 ) essential to start transcription in prokaryotes involved in the formation of transcription initiation complex
2. the - 35 region -25 bp upstream from the TATA box has the consensus sequence TTGACA important in initial recognition and binding of RNA polymerase to the DNA the first nucleotide transcribed is usually a purine.
Eukaryotic promoter
Eukaryotic promoters are extremely diverse and are difficult to characterize.
They typically lie upstream of the gene and can have regulatory elements several kilobases away from the transcriptional start site.
.
Many eukaryotic promoters also have a CAAT box with a GGNCAATCT consensus sequence centered at about -75.
Eukaryotic genes encoding proteins have promoter sites with a TATAAA consensus sequence, called a TATA box or Hogness box, centered at about - 25.
Promoters differ markedly in their efficacy
Genes with strong promoters are transcribed frequently as often as every 2 seconds in E. coli.
In contrast, genes with very weak promoters are transcribed about once in 10 minutes.
The -10 and -35 regions of most strong promoters have sequences that correspond closely to the consensus sequences
Weak promoters tend to have multiple substitutions at these sites.
Promoter element serve as binding site for trans-acting element (transcription factor or DNA-binding proteins that control gene expression)
have DNA-binding domain for interaction with promoters and an
activation domain to allow interaction with other transcription factors
The distance between these conserved sequences is also important; a separation of 17 nucleotides is optimal. Thus, the efficiency or strength of a promoter sequence serves to regulate transcription.
Mutation of a single base in either the -10 sequence or the -35 sequence can diminish promoter activity.
Enhancer element
Transcription of eukaryotic genes is stimulated by enhancer sequence, which can be quite distant (several kb) from the start site, on either its 5’ or 3’ side.
DNA between the enhancer and the promoter loops out to allow the proteins bound to the enhancer to interact directly either with one of the general transcription factors or with RNA polymerase itself.
Transcription begins when DNA- dependent RNA polymerase binds to promoter region and moves along the DNA to the transcription unit.
• In prokaryotes only single enzyme, RNA polymerase governs the synthesis of all cellular RNAs.
• There are 3 different eukaryotic RNA polymerases that are transcribed by 3 different sets of genes and are distinguished by their sensitivity to a fungal toxin α–amanitin :
• located in nucleolus • Synthesises rRNAs• Sensitive to α -amanitin
RNA polymerase I
• located in nucleoplasm• Synthesises snRNA• Very sensitive to α -amanitin
RNA polymerase I I
• located in nucleoplasm• Synthesises tRNA, 5SrRNA• Moderately sensitive to α -amanitin
RNA polymerase I I I
Structure of Prokaryotic RNA Polymerase
Upstream DNA
Downstream DNA
RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’ hydroxyl end, building RNA in the 5’ 3’ direction
RNA polymerase from E. coli is a very large (~400 kd) and complex enzyme consisting of four kinds of subunits: α2, β, β’, σ constituting an holoenzyme.
The σ subunit helps find a promoter site where transcription begins, participates in the initiation of RNA synthesis and then dissociates from the rest of the enzyme
RNA polymerase without this subunit (α2, β, β’) is called the
core enzyme contains catalytic site
Template
The template strand is the strand from which the RNA is actually transcribed. It is also termed as antisense strand.
The coding strand is the strand whose base sequence specifies the amino acid sequence of the encoded protein. It is also called as sense strand.
G C A G T A C A T G T C5' 3'
3' C G T C A T G T A C A G 5' template strand
coding strand
RNAG C A G U A C A U G U C5' 3'
transcription
Figure 7-9 Essential Cell Biology (© Garland Science 2010)
Promoter and terminator sequences of a gene tell the RNA polymerase where to start and stop transcription
‘the promoter site is encountered by a random walk in one dimension rather than in three dimensions’
The holoenzyme binds to duplex DNA and moves along
the double helix in search of a promoter, forming transient hydrogen bonds with exposed hydrogen-donor and -acceptor groups on the base pairs.
The search is rapid because RNA polymerase slides
along DNA instead of repeatedly binding and dissociating from it.
The σ subunit contributes to specific initiation in two ways:
1. It decreases the affinity of RNA polymerase for general regions of DNA by a factor of 104. In its absence, the core enzyme binds DNA indiscriminately and tightly.
2. The σ subunit enables RNA polymerase to recognize promoter sites.
A large fragment of a σ subunit was found to have an a helix on its surface; this helix has been implicated in recognizing the 5’-TATAAT sequence of the -10 region.
Different types sigma factor specific for sets of genes :
Sigma factor 70 (MW = 70 kDa) is most common form -initiates transcription at most promoters.
Sigma factor 32 (MW = 32 kDa) is produced after heat shock –initiates transcription at promoters of genes needed for responding to heat. Sigma factor 54 turns on genes for nitrogen utilization. Bacteriophage produces a powerful sigma factor that preferentially transcribes the phage DNA instead of the bacterial DNA.
Promoters of heat shock proteins
Products of this set of genes are made at higher levels when the cell receives a sudden
increase in temperature
RNA polymerase bind to the promoters of these genes only when σ70 is replaced with σ32 subunit, specific for heat shock promoters
RNA synthesis can start de novo, without the requirement for a primer.
RNA Polymerase unwind the template double helix for over a short distance, forming transcription ‘bubble’.
Elongation takes place at transcription bubbles that move along the DNA template.
RNA polymerase stays bound to its template until a termination signal is reached
The region containing RNA polymerase, DNA and nascent RNA is called a transcription bubble.
E. coli generally keeps about 17 bp unwind.
Elongation of transcript by E.coli RNA polymerase proceeds at a rate of 50-90 nucleotides/sec.
The RNA-DNA helix formed is about 8 bp long and corresponds to nearly one turn of a double helix.
The σ factor dissociates at random as polymerase enters the elongation phase.
The NusA protein binds to the elongating RNA polymerase, competitively with the σ subunit.
Once the transcription is complete, NusA dissociates from the enzyme, the RNA polymerase dissociates from the DNA and σ factor can now bind to the enzyme to initiate transcription.
Termination of RNA synthesis
1. Intrinsic Termination –
RNA transcript with self-complementary sequences
Permitting the formation the formation of hairpin loop structure
Centered 15-20 nucleotides before the projected end of the RNA
Highly conserved string of the A residues in the template strand are transcribed into U residues near the 3’ end of the hairpin.
Formation of hairpin structure in the RNA disrupts several A=U bp in the RNA-DNA hybrid segment
Disrupt interaction between RNA and the RNA polymerase
2. rho – dependent termination (-dependent termination). Lack the sequence of repeated A residues in the
template strand. Contain CA-rich sequence – rut (rho utilization
element).
ρ protein associates with the RNA at the binding sites and migrates in 5’-3’ direction (reaches transcription complex that is paused).
ρ factor has an ATP-dependent RNA-DNA helicase activity that promotes translocation of the protein along RNA and ATP is hydrolysed.
Control of Eukaryotic Gene Expression
Eukaryotes have more complex means to regulate gene expression because they have compartments (e.g., nucleus) within cells and multicellular structures that require cell differentiation.
Successful binding of active RNA polymerase II holoenzyme at one of its promoter usually requires the action of other proteins:
1. Transcription activators
2. Coactivators
3. Basal transcription factors
4. Chromatin modification and remodeling
Transcription activators
Mediate positive gene regulation. Binds to specific regulatory DNA sequences (e.g. enhancers) & enhance the RNA polymerase -promoter interaction.
It actively stimulates transcription.
Common in eukaryotes.
On binding to repressor, it brings confirmational changes which
leads to dissociation of repressor from the operator & increase in
transcription.
How do the activators function at a distance?The intervening DNA is looped so that the various protein complexes can interact directly.
Looping is promoted by certain nonhistone proteins that are
abundant in chromatin and bind nonspecifically to DNA
High mobility group (HMG)
play an important structural role in chromatin remodeling and transcriptional activation
Coactivator Protein ComplexesEukaryotic coactivator consists of 20 to 30 or more polypeptides in a protein complex called mediator.
Mediator binds tightly to the carboxyl-terminal domain (CTD) of largest subunit of Pol II.
Transcription activators interact with one or more components of the mediator complex.
Coactivator complexes function at or near the promoter’s TATA box.
ActivatorsDNA
Enhancer Distal controlelement
PromoterGene
TATA boxGeneraltranscriptionfactors
DNA-bendingprotein
Group of mediator proteins
RNApolymerase II
RNApolymerase II
RNA synthesisTranscriptioninitiation complex
Figure 18.10-3
What are Basal and Regulatory transcription factors? The basal transcription factors bind to the nearby
promoter region to the start site.
For example TATA element or initiator sequence.
They are the minimal complement of proteins necessary to reconstitute accurate transcription from a minimal promoter.
So the whole TFIIA,B,C,D,E,H,K family of factors
are basal transcription factors. They are the ones that bind in and out of the RNA polymerase region.
Regulatory transcription factors are TF factors that bind to sequences farther away from the initiation site.
Serve to modulate levels of transcription.
They are also called activators and bind to specific enhancer sequences way upstream from the start site.
Protein-DNA interaction
Regulatory proteins generally bind to specific DNA sequences.
Affinity is 104 to 106 times higher than their affinity for any other DNA sequences.
Typically, a protein-DNA interface consists of 10 to 20 contacts that involves different amino acids
each contributing to the binding energy of the protein-DNA interaction.
How the different base pairs in DNA can be recognized from their edges without the need to open the double helix?
The binding of a gene regulatory protein to the major groove of DNA
Most of chemical groups that differ among the four bases, permit discrimination between base pairs are hydrogen bond donor and acceptor groups exposed in major groove and most of the protein-DNA contacts that impart specificity are hydrogen bonds.
Within regulatory proteins, the amino acid side chains most often hydrogen-bonding to bases in the DNA are those of Asn, Gln, Glu, lys, arg residues.
Regulatory proteins possess DNA-binding motifs - Helix-Turn-Helix motif homeodomain motif - Zinc finger motif
Regulatory proteins having protein-protein interaction domains
- Leucine Zipper motif - Helix-Loop-Helix
Helix-Turn-Helix Motif
All of the proteins bind DNA as dimers in which the two copies of the recognition helix are separated by exactly one turn of the DNA helix (3.4 nm).
First DNA-binding protein motif identified
It comprises about 20 amino acids in two short α helices, each 7-9 amino acids residues long, separated by β-turn and is found in many proteins that regulate gene expression.
First identified in 3 prokaryotic proteins: two repressor proteins (Cro and cI) and the E. coli catabolite activator protein (CAP).
This structure generally is not stable by itself, it is simply the reactive portion of somewhat larger DNA-binding domain.
One of the 2 helices is recognition helix because it usually contains many of the amino acids that interact with the DNA in a sequence specific way.
When bound to DNA, the recognition helix is positioned in or nearly in the major groove.
The lambda repressor and Cro proteins control bacteriophage lambda gene expression, and the tryptophan repressor and the catabolite activator protein (CAP) control the expression of sets of E. coli genes.
Homeodomain motif
The homeodomain is folded into 3 alfa helices, packed tightly together by hydrophobic interactions (A) The part containing helix 2 and 3 closely resembles the helix-turn-helix motif, with the recognition helix (red) making important contacts with the major groove (B). The Asn of helix 3, for example, contacts an adenine. Nucleotide pairs are also contacted in the minor groove by a flexible arm attached to helix 1. The homeodomain shown here is from a yeast gene regulatory protein.
The homeodomain is a DNA-binding domain of 60 amino acids that has 3 α-helices.
The C-terminal α-helix-3 is 17 amino acids, binds in the major groove of DNA.
The N-terminal arm of the homeodomain projects into the minor groove of DNA.
Proteins containing homeodomains may be either activators or repressors of transcription i.e. they function as transciptional regulators, especially during eukaryotic development.
it was discovered in homeotic genes (genes that regulate the development of body patterns)
highly conserved, identified in proteins from wide variety of organisms including humans
Zinc finger motif
(A) The gene regulatory protein bound to a specific DNA site. This protein recognizes DNA using three zinc fingers of the Cys-Cys-His-His type arranged as direct repeats. (B) The three fingers have similar amino acid sequences and contact the DNA in similar ways. In both (A) and (B) the zinc atom in each finger is represented by a small sphere.
• About 30 amino acids residues form an elongated loop held together at the base by a single Zn2+ ion, coordinated to four of the 2 residues (four Cys, or two Cys and two His).
• Zinc does not itself interact with DNA.
• The coordination of Zinc with the amino acid residues stabilizes this small structural motif.
• The interaction of a single Zinc finger with DNA is typically weak.
• DNA-binding proteins like Zif268 have multiple Zinc fingers that enhance binding by interacting simultaneously with DNA.
Zinc finger domain exists in two forms:
1. C2H2 zinc finger: a loop of 12 amino acids anchored by two cysteine and two histidine residues that tetrahedrally co-ordinate a zinc ion.
This motif folds into a compact structure comprising two β-strands and one α-helix.
The α-helix containing conserved basic amino acids binds in the major groove of DNA.
Examples: (1) TFIIIA, the RNA Pol III transcription factor, with C2H2 zinc finger repeated 9 times. (2) SP1, with 3 copies of C2H2 zinc finger.Usually, three or more C2H2 zinc fingers are required for DNA binding.
2. C4 zinc finger: zinc ion is coordinated by 4 cysteine residues.
Example: Steriod hormone receptor transcription factors consisting of homo- or hetero-dimers, in which each monomer contains two C4 zinc finger.
Leucine zipper motif
• This motif is an amphipathic α-helix with a series of hydrophobic amino acid residues concentrated on one side the hydrobhobic surface forming an area of contact between the two
polypeptides of a dimer.
• Stricking feature : Leu residue at every seventh position form a straight chain along
the hydrophobic surface.
• Regulatory proteins with leucine zippers DNA binding domain with a high concentration of Lsy or Arg residues
interact with negatively charged phosphates of the DNA backbone.
• Leucine zippers have been found in many eukaryotic and a few bacterial proteins.
Helix-Loop-Helix
• Structural motif in eukaryotic regulatory proteins implicated in control of gene expression during the development of multicellular organisms.
• They share a conserved region of about 50 amino acid residues important in DNA binding and protein dimerization.
• Two short amphipathic α-helices linked by a loop of variable length.
• The Helix-Loop-Helix motifs of two polypeptides interact to form dimers.
• DNA binding is mediated by an adjacent short amino acid sequence rich in basic residues.
Eukaryotic gene expression is regulated at many stages Transcriptional ground state is restrictive – strong
promoters are inactive in vivo in absence of regulatory proteins.
In bacteria, it is nonrestrictive, RNA polymerase has access to every promoter , bind and initiate transcription at some level of efficiency in the absence of activators or repressor.
Distinguish the regulation of gene expression in eukaryotes from that in bacteria
1. Access to eukaryotic promoter is restricted by structure of chromatin.
2. Positive mechanisms predominate – transcriptional ground state is restrictive – every gene requires activation in order to transcribe.
3. Eukaryotic cells have larger, more complex multimeric regulatory proteins than do bacteria.
4. Transcription in the eukaryotic nucleus is separated from translation in cytoplasm in both space and time.
Transcriptionally active chromatin is structurally distinct from inactive chromatin
It is darkly stained region of the chromatin.
It is compactly coiled regions and with more DNA.
It is genetically inert as can not transcribe mRNA due to tight coiling.
It is late replicative.
About 10% of chromatin in typical eukaryotic cell is in more condensed form.
Associated with centromeres.
It is lightly stained region.
It is loosely coiled region and with less DNA.
It is genetically active.
It is early replicative.
Heterochromatin Euchromatin
Chromatin
Chromatin Remodeling
is the modification of chromatin architecture to allow access of condensed genomic DNA to the regulatory transcription machinery proteins, and thereby control gene expression.
Covalent histone modifications ATP-dependent chromatin remodeling complexes
Each of the core histones has two distinct structural domains:1. Central domain is involved in histone-histone interaction
and wrapping of DNA around the nucleosome.
2. Lysine rich amino-terminal domain – positioned exterior of nucleosome assembly.
Methylation of specific lysine residues in H3 and H4 causes condensation of DNA around histones preventing binding of transcription factors to the DNA leading to gene repression.
Methylation of lysines H3K4 and H3K36 is correlated with transcriptional activation while demethylation of H3K4 is correlated with silencing of the genomic region.
These methylations facilitates the binding of histone acetyltransferase (HATs) - acetyl groups are attached to positively charged lysines in histone tails (N-terminal).
This loosens chromatin structure, thereby promoting the initiation of transcription.
Histone
tailsDNA double helix
Nucleosome(end view)
Amino acidsavailablefor chemicalmodification
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones Acetylated histones(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Acetylation induces a conformational change in the core histones
Note: acetylation neutralizes the positive charge of lysine
HAT: Histone Acetyltransferase
DNA Methylation
• Methylation near gene promoters varies considerably depending on cell type, with more methylation of promoters correlating with low or no transcription (Suzuki & Bird, 2008).
• Proteins that bind to methylated DNA also form complexes with the proteins involved in deacetylation of histones.
• Therefore, when DNA is methylated, nearby histones are deacetylated, resulting in compounded inhibitory effects on transcription. Likewise, demethylated DNA does not attract deacetylating enzymes to the histones, allowing them to remain acetylated and more mobile, thus promoting transcription.
• In Neurospora crassa (Tamaru & Selker, 2001) and Arabidopsis thaliana (Jackson et al., 2002), H3-K9 methylation (methylation of a specific lysine residue in the histone H3) is required in order for DNA methylation to take place.
• When transcription of a gene is no longer required, the extent of acetylation of nucleosomes in that vicinity is reduced by the activity of histone deacetylation (HDACs).
• Methylation of lysines H3K9 and H3K27 is correlated with transcriptional repression (Rosenfeld et al., 2009 "Determination of enriched histone modifications in non-genic portions of the human genome." BMC Genomics 10: 143).
• H3K9 is highly correlated with constitutive heterochromatin (Hublitz et al., 2009) "Mechanisms of Transcriptional Repression by Histone Lysine Methylation" The International Journal of Developmental Biology (Basel) 10 (1387): 335–354).
In vertebrates and plants, many genes contain CpG islands near their promoters
1,000 to 2,000 nucleotides long
In housekeeping genes The CpG islands are unmethylatedGenes tend to be expressed in most cell types
In tissue-specific genesThe expression of these genes may be silenced by the
methylation of CpG islands
ATP-dependent chromatin remodeling complexes
The three best-characterized classes of ATP-dependent chromatin-
remodelling enzyme are the SWI/SNF, CHD (chromodomain and helicase-like domain) and ISWI (imitation SWI) families.
Each has a unique domain (bromo, chromo), known to interact with specific chromatin substrates.
NURF, member of ISW1 family, remodels chromatin in a way that complement and overlap the activity of SWI/SNF.
SWI/SNF complex contain bromodomain near the carboxy terminus of
active ATPase subunit, which interacts with acetylated histone tails.
SWI/SNF opens up DNA region where RNA Pol II, transcription factors and co-activators bind to turn on gene transcription. In the absence of SWI/SNF, nucleosomes can not move farther and remain tightly aligned to one another.
Additional methylation by methylase and deacetylation by HDAC proteins condenses DNA around histones, make DNA unavailable for binding by RNA Pol II and other activators, leading to gene silencing.
ATP-dependent chromatin-remodeling complexes regulate gene
expression by either moving, ejecting or restructuring nucleosomes.
These protein complexes have a common ATPase domain and energy from the hydrolysis of ATP allows these remodeling complexes to reposition (slide, twist or loop) nucleosomes along the DNA, expel histones away from DNA or facilitate exchange of histone variants, and thus creating nucleosome-free regions of DNA for gene activation (Wang et al., 2007 "Chromatin remodeling and cancer, Part II: ATP-dependent chromatin remodeling." Trends Mol Med. 13 (9): 373–80).
Examples of transcriptional regulation
1. Constitutive transcription factors: SP1
Binds to a GC-rich sequence with the consensus sequence GGGCGG.
Binding site is in the promoter of many housekeeping genes.
It is a constitutive transcription factor present in all cell types.
Contains three zinc finger motifs and two glutamine-rich activation domains.
2. Hormonal regulation: steroid hormone receptors
Many transcription factors are activated by hormones. Steroid hormones: lipid soluble and can diffuse through cell membranes to interact with transcription factors called steroid hormone receptors.
In the absence of steroid hormone, the receptor is bound to an inhibitor, located in the cytoplasm.
In the presence of steroid hormone,
the hormone binds to the receptor and releases the receptor from the inhibitor,
receptor dimerize and translocate to the nucleus.
receptor interacts with its specific DNA-binding sequence (response element) via its DNA-binding domain, activating the target gene.
Case Study
Positive and Negative Regulation of Transcription of the Yeast Galactose Utilization Genes
Three genes encode enzymes for metabolizing galactose
GAL1 encodes galactokinaseGAL7 encodes galactose transferaseGAL10 encodes galactose epimerase
Yeast cells have no operon - each of the GAL gene is transcribed separately.
In the absence of galactose, a Gal4p dimer binds the UASG (upstream activator sequence) along with the repressor protein Gal80p. No transcription occurs (quenching).
In the presence of galactose, Gal80p is bound to the inducer. A shift occurs, exposing the Gal4p activation domain; transcription proceeds
Gal4p – transactivatorGal80p – repressorGalactose – effector
Glucose is the preferred carbon source for yeast. When glucose is present, most of the GAL genes are repressed – whether galactose is present or not.
An operon is a cluster of bacterial genes along with an adjacent promoter that controls the transcription of those genes.
They usually control an important biochemical process. They are only found in prokaryotes.
Francois Jacob and Jacques Monod (1962) -first proposed the operon model of gene regulation
For their significant contribution in field of biochemistry, they were awarded Nobel Prize in Medicine in 1965.
The first system of gene regulation that was understood was the lac operon in E. coli.
Jacob, Monod & Lwoff
What are inducible genes ?
• They can be turned on or off – depending on the environment they are in.
• An Inducer acts as a ‘switch’ to turn the gene on or off.– a chemical substance in the nutrient medium
• The Inducer influences the transcription of the induciblegene(s) via controlling sites called Operators– on the DNA adjacent to the coding sequence of the gene(s).
• The Operator is usually where a regulatory protein binds.
General Organization of an Inducible Gene
General Organization of an Inducible Gene
Regulatory Proteins can activate or block transcription of inducible genes
The Lac operon - showing its genes and its binding sites.
The promoter is a specific DNA sequence to which the RNA Polymerase binds.
You can classify genes in a simple way in two classes
structural genes • are those that produce the enzyme required for lactose
metabolism
regulatory elements • determine whether transcription of the structural genes
will occur. They monitor and respond to environmental conditions (presence of lactose)
The structural genes and the regulatory elements form a functional genetic unit called the Lac Operon.
The lac operon
The lac operon consists of three genes each involved in processing the sugar lactose:
1. lacZ encodes β-galactosidase (LacZ), an intracellular enzyme that cleaves the disaccharide lactose into glucose and galactose.
2. lacY encodes β-galactoside permease (LacY), a membrane-bound transport protein that pumps lactose into the cell.
3. lacA encodes β-galactoside transacetylase (LacA), an enzyme that transfers an acetyl group from acetyl-CoA to β-galactosides.
The primary enzymatic function of β-galactosidase relevant to its role as a biotechnological tool is to cleave the chemical bond between the anomeric carbon and glycosyl oxygen of appropriate substrates (Serebriiskii & Golemis, 2000).
The lac operon regulatory elements are distributed along the DNA chain as (Reznikoff, 1992; Muller-Hill, 1998): the lac promoter is located between bp - 36 (relative to the starting point of gene lacZ, bp +1) and bp -7.
Prokaryotic genes are polycistron systems
All 3 genes of the lac operon are transcribed on the same messenger RNA.
Ribosomes translate the 3 proteins independently.
Unique feature of prokaryotes that is only very rarely seen in eukaryotes, where 1 gene per mRNA is the rule.
Adapting to the environment
• E. coli can use either glucose (monosaccharide) or lactose (disaccharide).
• Glucose needs to be hydrolysed (digested) first
So the bacterium prefers to use glucose when it can.
• It would be energetically wasteful for E. coli if the lac genes were expressed when lactose was not present.
• It achieves with the lac repressor which halts the production in the absence of lactose and EIIAGlc, which shuts down lactose permease when glucose is being transported into the cell.
Lac operon uses a two-part control mechanism
Diauxic dual control mechanism causes the sequential utilization of glucose and lactose in two distinct growth phases.
This phenomenon was originally studied by Monod (1941).
The first control mechanism is the regulatory response to lactose, which uses an intracellular regulatory protein called the lactose repressor to hinder production of β-galactosidase in the absence of lactose.
Diauxic growth curve
The existence of two different exponential growth phases, separated by a short interval in which the culture does not grow. The first phase corresponds to the bacterial culture feeding on glucose (lactose), while the interval with no growth corresponds to the time the bacteria need to turn on the genes needed to metabolize lactose after glucose exhaustion.
At low glucose concentrations, phosphorylated EIIA
accumulates and this activates membrane-bound adenylate cyclase.
Intracellular cyclic AMP levels rise and this then activates CAP (catabolite activator protein), which is involved in the catabolite repression system, also known as glucose effect.
When the glucose concentration is high, EIIA is mostly
dephosphorylated and this allows it to inhibit lactose permease.
When the genes in an operon are transcribed, a single mRNA is produced for all the genes in that operon. This mRNA is said to be polycistronic because it carries the information for more than one type of protein.
The regulatory gene lacI (constitutive) produces an mRNA that produces a Lac repressor protein, which can bind to the operator of the lac operon.
The Lac regulatory protein is called a repressor because it keeps RNA polymerase from
transcribing the structural genes
Thus the Lac repressor inhibits transcription of the
lac operon
Structure of Lac repressor
The binding sites for regulatory proteins are often inverted repeats of short DNA sequences (a palindrome) at which multiple (usually two) subunits of a regulatory protein bind.
The lac repressor is a homotetramer (dimer of dimers consisting of two functional homodimers of 37-kd subunits ) of lacI polypeptides (Lewis, 2005; Wilson et al., 2007).
An E. coli cell usually contains about 20 tetramers of the Lac repressor.
Each of the tethered dimers separately binds to a palindromic operator sequence, in contact with 17 bp of a 22 bp region in the lac operon.
Each dimer binds to a distinct DNA sequence at –82 and +11 respective to transcription start site.
This results in DNA looping, preventing the DNA polymerase from binding to –35 and –10 sequences.
In the absence of lactose, the Lac repressor binds to the operator and keeps RNA polymerase from transcribing the lac genes.
Effect of Lac repressor on the lac genes is referred to as negative regulation.
When lactose is present, the lac genes are expressed because allolactose binds to the Lac repressor protein and keeps it physically from binding to the lac operator.
In the "induced" state, the lac repressor is NOT bound to the operator.
Allolactose is an isomer of lactose. Small amounts of allolactose are formed when lactose enters E. coli.
Allolactose binds to an allosteric site on the repressor protein causing a conformational change
repressor can no longer bind to the operator region and falls off
RNA polymerase can then bind to the promoter and transcribe the lac genes
The nature of the lac inducer
Allolactose is called an inducer because it turns on or induces the expression of the lac genes.
The presence of lactose (and thus allolactose) determines whether or not the Lac repressor is bound to the operator.
When the enzymes encoded by the lac operon are produced, they break down lactose and allolactose, eventually releasing the repressor to stop additional synthesis of lac mRNA.
Whenever glucose is present, E. coli metabolizes it before using alternative energy sources such as lactose, arabinose, galactose, and maltose.
Glucose is the preferred and most frequently available energy source for E. coli.
The enzymes to metabolize glucose are made constantly by E. coli.
When both glucose and lactose are available, the genes for lactose metabolism are transcribed at low levels.
Sometimes the transport of glucose blocks the transport
of the inducer of the lac operon
Inducer exclusion External glucose decreases the efficiency of lac
permease to transport lactose (Reznikoff 1992), and by doing so negatively affects the induction of the operon genes.
Only when the supply of glucose has been exhausted does RNA polymerase start to transcribe the lac genes efficiently, which allows E. coli to metabolize lactose.
Maximal transcription of the lac operon occurs only when glucose is absent and lactose is present.
The action of cyclic AMP and a catabolite activator protein produce this effect.
Catabolite Activator Protein (CAP; also known as cAMP receptor protein, CRP) :
transcriptional activator, exists as a homodimer in solution, with each subunit comprising a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus
helix-turn-helix structure - binding is mediated by a helix-turn-helix motif in the protein’s DNA binding domain.
CAP binds a specific DNA site upstream from the lac promoter, and by doing so it increases the affinity of the RNA polymerase for this promoter (Reznikoff 1992). This regulatory mechanism is known as catabolite repression.
It binds to successive major grooves on DNA
this opens the DNA molecule up
allowing RNA polymerase to bind
transcribe the genes involved in lactose catabolism
Thus, CAP enhances the expression of the lac operon when lactose is present, but not glucose.
The presence or absence of glucose affects the lac operon by affecting the concentration of cyclic AMP.
The concentration of cyclic AMP in E. coli is inversely proportional to the concentration of glucose: as the concentration of glucose decreases, the concentration of cyclic AMP increases.
When the concentration of glucose is low, cAMP accumulates in the cell. The binding of cAMP and the catabolite activator protein to the lac promoter increases transcription by enhancing the binding of RNA polymerase to the lac promoter.
In the presence of lactose and absence of glucose, cyclic AMP (cAMP) joins with a catabolite activator protein that binds to the lac promoter and facilitates the transcription of the lac operon and stimulates RNA transcription 50 fold.
CAP-cAMP is a positive regulatory element responsive to glucose levels whereas the lac repressor is a negative regulatory element responsive to lactose.
The open complex of RNA polymerase and the promoter does not form readily unless CAP-cAMP is present.
CAP interacts directly with RNA polymerase through the polymerase’s α-subunit.
There are two ways of making a mutant strain where the lac operon is always on, regardless of whether lactose is present or not.
1. i- mutation: the lacI gene does not produce a functional repressor protein no repressor to bind to the operator RNA polymerase is never inhibited lac operon is always transcribed.
i- mutants are recessive: an i+ / i- heterozygote has normal gene regulation, because the wild type allele produces a normal repressor.
2. Many operator mutants are constitutive, oc
The operator is mutated so that the repressor can no longer bind to it. Transcription occurs and the lac operon is on when no repressor is bound to the operator.
trp operon The E. coli operon includes 5 genes, 7-kb mRNA
transcript for the enzymes required to convert chorismate to tryptophan.
Two of the enzymes catalyse more than one step in the pathway.
The mRNA from the try operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acids.
The trp operon is a repressible operon.
A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription.
Repression is associated with anabolic pathways -focus is on the "end products" of anabolic pathways.
Regulatory gene codes for a protein (the repressor) that
is inactive in absence of trp, repressor is inactive and RNA polymerase transcribes the structural genes.
The trp repressor is a homodimer, each subunit containing 107 amino acid residues.
A corepressor is a molecule that cooperates with a repressor protein to switch an operon off.
For example, E. coli can synthesize the amino acid tryptophan.
By default the trp operon is on and the genes for tryptophan synthesis are transcribed.
When tryptophan is present, it binds to the trp repressor protein, which turns the operon off. The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high.
Promoter
DNA
Regulatory gene
mRNA
trpR
5
3
Protein Inactive repressor
RNApolymerase
Promotertrp operon
Genes of operon
Operator
mRNA 5
Start codon Stop codon
trpE trpD trpC trpB trpA
E D C B A
Polypeptide subunits that make upenzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
(b) Tryptophan present, repressor active, operon off
DNA
mRNA
Protein
Tryptophan (corepressor)
Activerepressor
No RNAmade
Figure 18.3
The trp operator site overlaps the promoter, so binding of the repressor blocks binding of RNA polymerase. Repressor lowers transcription 70-fold (as compared
to derepressed state) attentuation permits another 10-fold control total dynamic range of control = 700-fold.
The regulation of the trp operon is achieved by means of two mechanisms controlling successive stages of expression:
1. Repression regulates initiation of transcription by blocking attachment of RNAP.
2. Attenuation is responsible for premature termination of transcription due to conformational changes in nascent mRNA.
Attenuation in the control of expression of bacterial operons (Yanofsky C, 1981 Nature 289(5800):751-8).
…
…
Attenuator Region of Trp Operon
Low tryptophan: transcription of trp operon genes RNA polymerase reads through attenuator.
High tryptophan: attenuation, premature termination attenuator causes premature termination of transcription. The terminator consists of an inverted repeat followed by string of eight A-T pairs. The inverted repeat forms a hairpin loop.
When RNA polymerase reaches string of U’s, the polymerase pauses, the hairpin forms Transcript is released
Termination occurs before transcription reaches the trp (structural) genes.preventing hairpin formation would destroy termination signal transcription would proceed
Mechanism of Attenuation The trp operon attenuation mechanism uses signals
encoded in four sequences within 162 nucleotide leader region at the 5’ end of the mRNA, preceding the initiation codon of the first codon.
Within the leader lies a region Attenuator, made up of sequences 3 and 4
base pair to form a G C rich stem-and-loop structure closely followed by a series of U residues
attenuation sequence acts as a transcription terminator
sequence 2 is a alternative complement for sequence 3
Leader sequence
mRNA produced from attenuator region can fold into two different secondary structures
Stem loops: 1-2, 3-4 Stem loop: 2-3
Formation of stem loop structures; 1-2 and 3-4 is more stable and results in the formation of a termination (hairpin loop) structure/signal.
Formation of stem loop structure 2-3 would result in the disruption of stem loops 1-2/3-4.
The stem loop structure formed between 2-3 does not result in termination signal attenuator structure cannot form, transcription would proceed.
Posttranscriptional Regulation
Control of gene expression usually involves the control of transcription initiation.
But gene expression can be controlled after transcription, with mechanisms such as:
– RNA editing– RNA interference– alternative splicing (different mRNA molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns)– mRNA degradation
RNA editing
Some mRNAs are edited before translation.
RNA editing has been observed in mRNA, tRNA and rRNA.
It has been detected in mitochondria and chloroplasts and in nuclear encoded RNAs but as yet not in procaryotes.
RNA editing can be divided into two categories.
1. Insertion/Deletion RNA editing- nucleotides are inserted or deleted as occurs in mitochondrial mRNAs of trypanosomes, a kind of protozoan.
The initial transcripts of the genes that encode cytochrome oxidase subunit II in some protist mitochondria provide an example of editing by insertion.
These do not correspond precisely to the sequence needed at the carboxyl terminus of the protein product.
A number of U nucleotides are inserted or deleted to create the translatable mRNAs.
Specific small RNAs, guide RNAs interact with the mRNA to define the position of editing.
Base pairing between the initial transcript and the guide RNA involves a number of G=U base pairs, common in RNA molecules.
2. Nucleotides are modified to change one nucleotide into another. One example is the de-amination of cytidine which occurs in mammalian apolipoprotein B mRNA in the intestine.
Here, a specific C is changed into a U, introducing a stop codon for translation and thereby a shorter version of the protein.
The cytidine deaminations are carried out by the apoB mRNA editing catalytic peptide family of enzymes (APOBEC).
A second example is de-amination of adenosine to inosine.
Inosine is read as guanosine by the translation machinery.
The adenosine is present in a double stranded region of the mRNA and the enzyme adenosine de-aminase that acts on RNA (ADAR) catalyzes the reaction.
It is common in transcripts derived from the genes of primates (90% or more of the editing occurs in the short interspersed elements (SINES) called Alu elements).
Concentrated near protein-encoding genes, often appearing in introns and untranslated regions at the 3’ and 5’ ends of transcripts.
The ADAR enzymes bind to and promote A-to-I editing only in duplex regions of RNA.
Defects in ADAR function have been associated with a variety of human neurological conditions, including amyotrophic lateral sclerosis (ALS), epilepsy, and major depression.
dsRNA as a regulator of gene expression
dsRNA has role in several chromatin and/ or genomic DNA modifications, which lead in the regulation of specific genes.
dsRNA dependent mechanism can act at both transcriptional as well as post transcriptional levels.
This type of gene expression is given different names in different organisms.▫ RNA interference (RNAi) , in case of animals.▫ Post transcriptional gene silencing (PTGS) , in case of
plants.▫ Quelling, in case of filamentous fungi.
Inhibitors of gene expression
Rifamycin
Streptovarcins Streptolydigin
they tightly bind with β-subunit of RNA polymerase (rpoB) and inhibit initiation and transcription
Transcriptional regulation network of cold-responsive genes in higher plants
(Tongwen Yang, Lijing Zhang , Tengguo Zhang , Hua Zhang , Shijian Xu , Lizhe An, 2005; Plant science 169 (2005) 987–995).
• ABA, abscisic acid; • CBF, C-repeat-binding factor; • COR, cold-regulated; CRT, C-repeat; DRE,• dehydration-responsive element; • DREB, DRE-binding factor; DREB, • DRE-binding protein; • ICE, inducer of CBF expression;
CBF (DREB1) genes act as nodes of regulatory network in Arabidopsis response to cold stress.
With the use of genetic and molecular approaches, a series of regulatory genes involved in CBF cold response pathway have been isolated and analyzed.
Many plants increase freezing tolerance in response to low, nonfreezing temperature, a phenomenon known as cold acclimation.
With Arabidopsis as model plant, many cold response genes were isolated are of CBF family key components in transcriptional regulation of cold-responsive genes.
The expression of CBFs in Arabidopsis is also regulated by ABA, light and the circadian clock.
Arabidopsis encodes a small family of cold-responsive transcriptional activators known either as CBF1, CBF2, and CBF3 or DREB1b, DREB1c, and DREB1a.
The CBF transcription factors are members of the AP2/EREBP family of DNA-binding proteins, recognize the cold- and dehydration-responsive DNA regulatory element designated the CRT/DRE.
Have a conserved 5 bp core sequence of CCGAC, are present in the promoter regions of many cold- and dehydration-responsive genes of Arabidopsis, including COR.
Multiple biochemical changes that are associated with cold acclimation and thought to contribute to increased freezing tolerance, occur in non-acclimated transgenic Arabidopsis plants that constitutively express CBF3.
The homologous components of the Arabidopsis CBF cold response pathway have been found in many plants, including soybean, broccoli, alfalfa, tobacco, wheat, corn, rice and barley.
These CBF-like proteins from different species not only have high conserved 60 amino acid AP2/EREBP DNA-binding domain but also present conserved amino acid sequences.
Constitutive over-expression of the Arabidopsis CBF genes in other plants resulted in increase freezing tolerance.
Many cold-regulated genes of Arabidopsis are inducible by ABA as well as by cold.
This occurs via two separate signaling pathways, the ABA-dependent pathway and ABA-independent pathway.
DREB2 plays a role in drought adaptation in an ABA-independent manner.
Later three other CBF-related genes in Arabidopsis have been identified DREB1D, E, and F.
The DREB1D (CBF4) have been demonstrated to be inducible by ABA and drought but not by cold.
CBF1-3 transcript levels also increase in response to elevated ABA levels and suggest that both the cold-inducible CBF transcriptional factors and the non-cold-inducible CBF4 could be involved in activation of the CRT/DRE by ABA.
Cold-induced gene expression through CRT/DRE is greatly enhanced by light signaling, in which phytochrome B is required.
Light enhanced the induction kinetics of CBF1-3 encoding the transcription factors in a consecutive manner compared to the dark condition in the cold
suggest that the connection between cold and light signaling mediated by phytochrome is at higher step than the expression of CBF gene.
The circadian clock also gates expression of the CBF1-3 genes in response to low temperature in Arabidopsis.
CBF3 transcripts accumulate to maximum levels in the early morning and reach minimum levels in the early evening in Arabidopsis grown on a 12 h photoperiod.
Transcriptome studies suggest a diversity and complexity of the cold response pathways.
By microarrays technology and transcriptional profiling analysis approach, 8000 genes were determined at multiple times after plants were transferred from warm to cold temperature and in warm grown plants that constitutively expressed CBF1, CBF2, or CBF3.
These results indicate that extensive down regulation of gene expression occurs during cold acclimation.
CBF expression at warm temperatures repressed the expression of eight genes that also were down-regulated by low temperature, indicating that in addition to gene induction, gene repression is likely to play an integral role in cold acclimation.
Potential application of regulatory factors in crop anti-freezing engineering
Understanding the molecular mechanisms that plants have evolved to tolerate environmental stresses has the potential to provide new tools and strategies to improve the environmental stress tolerance of crops.
Since freezing tolerance is a multigenic trait, transformation of a single functional gene appears to have a limited effect on crop freezing tolerance.
Because many aspects of cold adaptation process are under transcriptional control, many transcription regulatory factors were chosen as one of the best targets for engineering crops to achieve enhanced cold tolerance.
Constitutive over expression of the CBF genes using the cauliflower mosaic virus 35S promoter can result in undesirable agronomic traits.
In Arabidopsis, CBF over expression can cause a ‘‘stunted’’ growth phenotype, a decrease in seed yield and a delay in flowering.
Using stress-inducible or artificial cold-inducible promoters may be a ideal approach to improve cold tolerance without causing negative agronomic effects.
Though many transcription regulatory factors were cloned and identified, only CBF genes have been successfully used to engineering cold stress tolerance in several species.
RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation(Gama-Castro et.al., 2008; Nucleic Acids Research)
RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database offering curated knowledge of the transcriptional regulatory network of Escherichia coli K12, currently the best-known electronically encoded database of the genetic regulatory network of any free-living organism.
RegulonDB contains detailed information of the different elements that conform the known regulatory network of the cell, such as transcription factors (TFs), small RNAs (sRNAs) and operon structures with their associated regulatory elements: promoters, TF binding sites and terminators.
RegulonDB is complemented with computational analyses and genome wide predictions of operons, promoters, TF binding sites, ribosome-binding sites and, for the first time, RNA regulatory target sites.
Visualizing tools in RegulonDB allow the user to navigate in the genome (Genome browser), to identify co-regulators for a particular TF, to locate the genes’ immediate neighbors in the regulatory network, and to identify sets of genes predicted to be functionally related (Nebulon tool).
Moreover, it also incorporates tools for the analysis of the transcriptional regulation of global gene expression experiments made in E. coli K12 (GET tools), as well as for exhaustive analyses focused on the detection of regulatory signals in upstream regulatory regions (RSA tools).
RegulonDB is mainly a manual database of regulatory information in E. coli incorporated by a team of curators from the primary literature.
PubMed abstracts are selected using a set of pertinent key words related to gene regulation.
When there is direct or suspected new relevant information, the full text of the articles is analyzed and the data are added to RegulonDB.
Starting on January 2008, every release of RegulonDB and EcoCyc will contain up-to-date curation with a delay of no more than 3 months.
The evidences associated to all RegulonDB objects are now classified as ‘strong’ or ‘weak,’ based on the confidence level of the experiment.
The evidences associated to all RegulonDB objects are now classified as ‘strong’ or ‘weak,’ based on the confidence level of the experiment.
Examples of strong evidences are DNA binding of purified TF for regulatory interactions, mapping of TSSs for promoters, and length of mRNA for transcription units.
On the other hand, gene expression analyses and computational predictions are considered weak evidences.
Generation of computational predictions for four different promoters of the σ70 family: those of σ 24, 28, 32 and 38.
The putative +1 of transcription initiation along with the -35 and -10 boxes can be downloaded from RegulonDB.
The active and inactive conformation of TFs is regulated by specific cell signals (‘effectors’) that can be metabolites, ions or other chemical-signaling molecules, through covalent or allosteric interactions.
The origin of these effectors can be endogenous (synthesized inside the cell), exogenous (incorporated or transported from outside the cell) or both (hybrid).
This feature has been added to the TFs in the database and a link to a specific web page that shows details of the cell sensing properties of the transcriptional regulators has been created.
RegulonDB v.6.0 has an expanded conceptual and relational model that includes other levels and mechanisms of regulation of gene expression, such as transcriptional elongation, posttranscriptional modification and translational initiation.
The first elements that are now modeled and populated are RNA regulatory elements, specifically riboswitches and attenuators, and small RNAs.
The user interface has a graphic representation and textual information about their sequences, location, evidences and references.
Riboswitches and attenuators are cis-regulatory elements that can modulate transcription elongation or translation initiation.
A riboswitch is part of the 50 non-translated region in specific bacterial mRNAs that can modulate gene expression in direct response to small molecules without the need for a protein intermediate.
These regulatory elements are highly conserved, both in structure and sequence, due to the constraints of forming a highly structured binding pocket for the effector.
Riboswitches are usually found associated with transcription or translation attenuators.
Several of these riboswitches have been experimentally described and their sequences are obtained from Rfam, a database of RNA families.
Attenuators are segments of RNA in the untranslated regions of some mRNAs that can form several mutually exclusive secondary structures, contrary to riboswitches, are rarely conserved at the sequence level.
The sRNAs genes code for RNA sequences of <350 nucleotides long can have intrinsic catalytic activity, modify a protein activity.
RegulonDB includes 49 interactions between sRNAs and their target genes.
RegulonDB literature can now be searched with the Textpresso text-mining engine, customized for E. coli.
Textpresso allows direct exploration of the curated literature, both at the level of highly specific key words and with entire categories.
The user can, for example, search for a type of regulation in which a gene or operon and a specific TF are mentioned within sentences of different papers.
The tool can search through 2472 full-text papers, 3125 paper abstracts, and more than 4200 curator notes.
The addition of this text-mining tool to RegulonDB will expand the possibilities, for the end user, of traversing the knowledge space of E. coli metabolism and gene regulation and will allow our curators to refine and confirm their annotations.
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