Regulation of Gene Expression at the level of Transcription (examples are mostly bacterial) Diarmaid Hughes ICM/Microbiology VT2009
Regulation of Gene Expression at the level of Transcription (examples are mostly bacterial) Local regulation (regulation of individual genes or operons) At what level does regulation occur? (i) Initiation of transcription Negative: Positive: Reducing polymerase access to a promoter Protein Repressors, etc. Increasing polymerase access to a promoter Protein Activators, etc. (ii) Termination of transcription Attenuation (early termination of transcription elongation) How? In response to what signals? Antitermination (continued transcription through terminators) How? In response to what signals? Riboswitches (one of the mechanisms) Often a gene is regulated by several mechanisms
Basic mechanisms of regulation of Transcription Initiation a. Absence of regulatory Proteins b. Repressor prevents polymerase binding c. Activator enhances polymerase binding
Basic mechanisms of regulation of Transcription Initiation Some activators work by Allostery Activator in this example promotes the transition to an open (active) polymerase comformation
Basic mechanisms of regulation of Transcription Initiation Action at a Distance and DNA Looping are often important in regulating gene expression. Some proteins interact with each other even when bound to sites well separated on the DNA
Basic mechanisms of regulation of Transcription Initiation DNA-bending protein can facilitate interaction between DNAbinding proteins at a distance
Control at the level of transcription initiation: the lac operon: 1 regulatory gene, 3 stuctural genes, and 2 control elements lacz encodes β-galactosidase (lactose hydrolysis) lacy encodes lactose permease (transports lactose across the cell wall) laca encodes thiogalactoside transacetylase (gets rid of the toxic thiogalacosides) A repressor and an activator together control the lac genes The repressor: lac repressor encoded by laci gene; responds to lactose. The activator: CAP (Catabolite Activator Protein) also called CRP (camp Receptor Protein); responds to the glucose level (which affects the level of cyclic AMP, camp). The enzymes required for the use of lactose as a carbon source are only synthesized when lactose is available as the sole carbon source.
lac operon The site bound by lac repressor protein is called the lac operator. The lac operator overlaps promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter.
lac operon Lac repressor binds as a tetramer, with each operator is contacted by a repressor dimer. The lac repressor protein has 2 states: it can either bind to lactose (technically, to a lactose derivative called allolactose) In complex with lactose it floats freely in ther cytoplasm RNA polymerase can bind the promoter The operon is transcribed makes products to use lactose In absence of lactose the repressor can bind to the operator region of the lac operon. This prevents the RNA polymerase from binding to the promoter The operon is not transcribed. Logic: if no lactose present, no need to make the enzymes to use it! If lactose appears, the operon is said to be induced. The lactose binds to the repressor, which then falls off the operator and allows transcription to occur.
lac operon CAP/CRP and catabolite repression The lac operon is negatively regulated by the lac repressor Prevents transcription initiation unless an inducer (lactose) is present. The lac operon is also positively regulated by CAP (catabolite activator protein, also called CRP) in complex with the small molecule cyclic AMP (camp). Logic: E. coli would prefer to use glucose as its food source. In the presence of glucose, the lac operon (and other similar genes) are turned off, even if lactose is present in the medium. This phenomenon is called catabolite repression. camp is made from ATP. When the glucose level in the cell is high, the camp level is low, because glucose inhibits synthesis of camp. When the glucose level is low, the camp level is increases. camp combines with the CAP protein to form a complex that binds to part of the lac operon promoter. This complex bends the DNA in a way that makes it much easier for RNA polymerase to bind to the promoter. This allows transcription to occur, but only if the lac repressor isn t present. This is positive regulation because the binding of CAP to the DNA causes transcription to occur.
lac operon CAP (with camp) binds to a site with a similar structure to the operator, 60 bp upstream of the start site of transcription. CAP interacts with the α-subunit of polymerase and recruits it to the promoter. Diauxic growth Diauxic growth curve: use best carbon source first (glucose), then switch over to the next (lactose)
Take home message To activate the lac operon two conditions must be met: 1. Absence of glucose (causes high camp, and activation of CAP) 2. Presence of Lactose (causes inactivation of the lac repressor) The absence of glucose signals the potential activation of genes capable of making proteins that can break down alternative carbon sources. The presence of Lactose signals that the lac operon should be activated. Put your foot on the accelerator, remove your other foot from the brake.
Variations in the details of CAP activation of promoters CAP is activated by binding of camp (intracellular levels elevated when glucose is low) CAP Protein dimerizes and binds to DNA (promoter region) binding induces a high angle bend in DNA Class I CAP binding sites can be from 62 to 103. CAP interacts with the carboxy terminus of the RNAP α- subunit (αctd) Class II CAP binding sites usually overlap the 35. CAP interact with the αctd, αntd, and the σ factor CAP contacts with RNAP stimulate transcription
Allosteric activation: example ntrc The majority of activators work by recruitment, such as CAP. These activators simply bring an active form of RNA polymerase to the promoter In this case of allosteric activation, RNAP initially binds the promoter in an inactive complex, and the activator triggers an allosteric change in that complex to activate transcription. Low nitrogen levels NtrC phosphorylation and conformational change The DNA binding domain binds DNA sites at ~ -150 position NtrC interacts with σ 54 (glna promoter recognition) ATP hydrolysis and conformation change in polymerase transcription STARTs
Allosteric activation: example merc MerR activates transcription by twisting promoter DNA MerR controls a gene called mert, (encodes an enzyme that makes cells resistant to toxic effects of mercury) In presence of mercury, MerR binds to a sequence between 10 and 35 regions of the mert promoter and activates mert expression. When Hg2+ is absent, MerR binds to the promoter and locks it in the unfavorable conformation When Hg2+ is present, MerR binds Hg2+ and undergo conformational change, which twists the promoter to restore it to the structure close to a strong σ70 promoter
arabad operon AraC and CAP control the arabad operon The promoter of the E. coli arabad operon is activated in the presence of arabinose and the absence of glucose and directs expression of genes encoding enzymes required for arabinose metabolism. Different from the Lac operon, two activators AraC and CAP work together to activate the 194 bp arabad operon expression CAP site Different from the Lac operon: Two activators, AraC and CAP, work together to activate the arabad operon expression
Variations on a theme Repressors can impart different regulatory patterns Arginine biosynthesis Lactose degradation Repression Induction Brock Biology of Microorganisms, vol. 9, Chapter 7
Variations on a theme A co-repression mechanism arginine biosynthesis Example: arginine, one of 20 essential amino acids. Bacteria can make their own, but if supplied with arginine will not express the biosynthetic gene (Why bother making it if there is plenty around?) No arginine Cell needs it Repressor does not bind Arginine genes expressed High arginine Cell does not need it Repressor + Arg bind Arginine genes not expressed Arginine Brock Biology of Microorganisms, vol. 9, Chapter 7
Variations on a theme An inducible repressor mechanism lactose metabolism Example: lactose, sugar carbon (food) source. Bacteria will utilize lactose if it is present, but do not express genes if no lactose is around (Why bother to make enzymes for using lactose, if it is not present?) No lactose Cell cannot use it Repressor binds to lac genes No Lac enzymes are produced Lactose present Cell can use it as nutrient Repressor + lactose fall off DNA Lac enzymes are made Lactose Brock Biology of Microorganisms, vol. 9, Chapter 7
Variations on a theme Transcriptional activation mechanism maltose metabolism Example: maltose, sugar carbon (food) source bacteria utilize maltose if present, but do not express genes if no maltose. (Why bother to make enzymes for using if it is not present?) No maltose Activator does not bind DNA or polymerase No Mal enzymes are produced Maltose present potential nutrient Activator + Maltose, bind polymerase to DNA Mal enzymes are made Promoter is weak Maltose Brock Biology of Microorganisms, vol. 9, Chapter 7
Part 2 Gene regulation AT or AFTER transcription initiation: the tryptophan operon Amino acid biosynthetic operons are usually controlled by premature transcription termination The trpoperonin E. coli encodes five structural genes required for tryptophan synthesis. These genes are regulated to efficiently express only when tryptophan is limiting. Two levels of regulation are involved: (1) Transcription repression by the Trp repressor (at initiation); (2) Attenuation (premature transcription termination) In response to the level of charged Trp-tRNA (a measure of the requirement for additional Trp synthesis)
The TRP operon The Trp repressor Trp repressor is encoded from trpr in a separate operon, and specifically interacts with the operator that overlaps with the promoter sequence. However, the repressor can only bind to the operator when it is complexed with tryptophan. Therefore, the amino acid Trp is a co-repressor and inhibits its own synthesis through end-product inhibition (negative feed-back regulation). This is just like the arginine example shown earlier. In contrast: lactose acts as an inducer of the lac operon
The TRP operon Trp Attenuation Transcription of the trp operon is prematurally stopped if the tryptophan level is not low enough. Results in the production of a leader RNA of 161 nt. Low Trp level High Trp level Trp Repressor: the primary switch to regulate expression of trp operon Trp Attenuation: a fine regulatory switch
The TRP operon Trp Attenuation 1. Transcription and translation in bacteria are coupled. Synthesis of the leader peptide follows immediately transcription of leader RNA. 2. The leader peptide contains two tryptophan codons. If tryptophan level is low, there will be little charged Trp-tRNA, and ribosome will pause at these sites. 3. Ribosome pausing at these sites alters the secondary structure of the leader RNA, (eliminates an intrinsic terminator structure) and facilitates transcription of the trp operon to continue. Terminator Attenuate transcription Ribosome pause Continue transcription of operon
The TRP operon Trp Attenuation Importance of attenuation 1. A typical negative feed-back regulation 2. Use of both repression and attenuation allows a fine tuning of the level of the intracellular tryptophan. 3. Attenuation alone can provide robust regulation: other amino acids operons like his and leu have no repressors and rely entirely on attenuation for their regulation. 4. An example of regulation using RNA structure (see riboswitches later). Attenuation in other operons
Regulation of the Trp operon in Bacillus subtilis 1. OVERVIEW: Transcription of the trp operon is regulated by transcription attenuation, in response to the availability of tryptophan, and charged trnatrp. BUT, the mechanism is different to E. coli. TRAP mechanism of regulation HIGH TRP: Tryptophan activates an RNA-binding protein, TRAP, which regulates transcription termination in the leader region of the trp operon (associated with a programmed transcription pause). ANTI-TRAP mechanism of regulation LOW TRP: Uncharged trnatrp activates transcription and translation of the at operon, leading to the synthesis of the anti-trap protein, AT. AT binds to TRAP and prevents TRAP from functioning. T-BOX mechanism of regulation 4. The production of AT is regulated by the binding of uncharged trnatrp to a leader transcript in the at operon. This stabilizes an antiterminator structure in the RNA. TRAP = trp RNA-binding Attenuation Protein AT = Anti-TRAP T-box = trna-binding sequence in RNA leader
Regulation of the Trp operon in Bacillus subtilis TRAP mechanism During transcription, RNA polymerase pauses following addition of U107. Under tryptophan-limiting conditions, formation of the antiterminator promotes transcription readthrough into the trp operon structural genes. In the presence of tryptophan, TRAP binds to the (G/U)AG repeats, thereby releasing the paused RNA polymerase and preventing antiterminator formation. As transcription proceeds, formation of the overlapping terminator causes transcription termination following synthesis of G140 or U141.
Regulation of AT (anti-trap) by T-Box mechanism ycaa-ycbk operon Overexpression of the protein encoded by ycaa increases trp operon expression Product of ycaa is AT antagonist of TRAP Promoter Leader ycaa ycbk ycaa transcription is regulated by binding of uncharged trna Trp to the leader/t-box! Binding of uncharged trna Trp affects the structure of the leader resulting in the formation of an antiterminator structure
How a T-Box mechanism works This is just an example and has no direct connection totrp operon regulation This is an example of a riboswitch (ligand binding determines a regulatory RNA conformation)
Summary of antitermination regulation mechanisms TRAP ANTI-TRAP T-Box Molecular mechanisms used in regulating transcription of the tryptophan biosynthetic operon of Gram-positive bacteria. (a c) trp operon regulation in B. subtilis and its closest relatives. In these organisms transcription of the operon is believed to be regulated by transcription attenuation solely, and is based on l-trp activation of an RNA-binding protein, TRAP. (a) When cells have a low intracellular level of l-trp, the TRAP protein is inactive, and the antiterminator structure forms in the transcript of the leader region of the trp operon. This structure prevents formation of the terminator structure, permitting transcription to proceed into the structural gene region. (b) When there is excess of L-Trp, TRAP is activated, and it binds to the leader RNA segment of the trp operon transcript. This prevents formation of, or disrupts, the antiterminator structure, enabling the RNA transcription terminator structure to form, and terminate operon transcription. (c) A second regulatory protein, AT, encoded in the at operon, can bind to Trp-activated TRAP and prevent the latter from binding to RNA and regulating trp operon transcription. AT synthesis is regulated transcriptionally by the T box mechanism, in response to uncharged trnatrp, and regulated translationally, also by trnatrp, during synthesis of a Trp-containing leader peptide. (d,e) trp operon regulation in other Firmicutes. In most Firmicutes, the trp biosynthetic operon is regulated by the T-box riboswitch mechanism. This regulatory mechanism is based on the formation of conserved RNA secondary structures capable of recognizing uncharged trnatrp. (d) Uncharged trnatrp stabilizes an RNA antiterminator structure, preventing transcription termination, and promoting transcription of the remaining segment of the operon. (e) When the pool of charged trnatrp is elevated, this charged trna is incapable of stabilizing the antiterminator structure, enabling the transcription terminator to form, and terminate transcription. From Trends in Genetics (2007) 23:422-426, Gutiérrez-Preciado, Janofsky, and Merino.
Bioinformatics: different regulatory mechanism used in different bacteria Operon organization and evolutionary history of the genes of the tryptophan biosynthetic pathway in Firmicutes.
Transcription Antitermination by proteins interacting with RNA polymerase. Example: Antitermination in bacteriophage λ RNA polymerase that transcribes through the nut region of λ becomes termination resistant.
Riboswitch Regulation of Gene Expression Riboswitches are regulatory RNA elements (structures) present in untranslated regions of mrnas. They act as direct sensors of small molecule metabolites and control gene transcription or translation in response to binding these small molecule metabolites. Their purpose is to regulate gene expression. Some operate at the level of transcription termination Others operate at the level of translation Another kind responds to the uncharged trna rather than responding to a metabolite. Others respond to temperature (no ligand involved).
Riboswitches 5 UTR 3 UTR 5 Aptamer Coding section 3 RNA control elements that regulates gene expression, without the participation of proteins Utilize a unique mechanism where by small molecules bind to aptamer/box region causing a conformational switch Were found initially in 5 UTR of bacteria with successive discoveries in prokaryotes
Riboswitches Riboswitch Ligands There are several confirmed riboswitches with unique metabolite ligands. Many others are under investigation. Figure shows the chemical structures of riboswitch ligands and schematics of conserved secondary structure in riboswitches.
Riboswitches riboflavin transporter mechanism (Flavin mononucleotide) Transcription attenuation Translation attenuation
Riboswitches Riboswitches are an important mechanism of gene regulation. For example, nearly 2% of the genes of the model organism Bacillus subtilis appear to be controlled by riboswitches. Riboswitches demonstrate that naturally occurring RNA can bind small molecules specifically, a capability that many previously believed was the domain of proteins. The existence of riboswitches in all domains of life therefore adds some support to the RNA world hypothesis, which holds that life originally existed using only RNA, and proteins came later; this hypothesis requires that all critical functions performed by proteins could be performed by RNA.