Regulation of Prokaryotic Transcription I. OVERVIEW A. Anabolic enzyme encoding genes 1. Code for enzymes that act in building molecules that are essential for survival 2. These genes are transcribed unless their end-product they need is present in the growth medium B. Catabolic enzyme encoding genes 1. Code for enzymes that break down molecules for energy 2. These genes are transcribed when the molecule to be degraded in present and turned off if there are no molecules to break down II.TERMINOLOGY A. Operons can genes, a promoter, and an operator 1. A cluster of related genes a) An advantage of arranging genes with related functions together is that only the control of their transcription can occur at just one point (1) For example, if bacteria needs to make histidine, all of the genes for this pathway are transcribed at once 2. A promoter a) A sequence of nucleotides in DNA to which RNA polymerase binds b) The sequence is usually located upstream from the genes (1) Often involves the –10 TATAAT box and a -35 region 3. An operator a) A segment of DNA to which regulatory proteins bind b) Location varies and is sometimes upstream from the promoter or sometimes downstream c) Controls the binding of RNA polymerase to its promoter B. Types of regulation 1. Overview of regulation in prokaryotes a) Catabolic pathways (1) Enzymes used to degrade carbon sources are only desired when those carbon sources are present b) Biosynthetic pathways (1) Enzymes used to construct biosynthetic building blocks are desired only if those building blocks are in low concentration (2) This prevents wasting energy on biosynthesis of enzymes and building block when there is a sufficient external supply 2. Negative control a) A regulatory protein, called a repressor protein, binds to the operator (1) The repressor molecule prevents RNA polymerase from binding properly to the promoter sequence b) Repressible (1) The repressor protein is synthesized in an inactive form (a) By inactive, I mean it cannot block RNA polymerase binding to the promoter (2) The repressor is activated by the binding of a corepressor (a) By active, I mean the repressor prevents RNA polymerase binding to the promoter (3) This type of control is seen most often in anabolic pathways (e.g., arginine biosynthesis) (a) The corepressor is often the end product of the anabolic pathway c) Inducible (1) The repressor protein is synthesized in an active form (a) Binds to the operator and prevents RNA polymerase from binding to promoter (2) When an inducer binds to the repressor, the repressor becomes inactive (a) RNA polymerase is now free to initiate transcription (3) This type of control is seen most often in catabolic pathways (e.g., lactose catabolism) (a) The inducer is often a form of the substrate that will be degraded 3. Positive control a) A regulatory protein, called an activator protein, binds to the operator (1) The activator molecular stabilizes RNA polymerase binding to the promoter region (a) Example: the arabinose catabolism 4. Regulatory proteins (for both positive and negative regulation) a) These are encoded by regulatory genes b) These are synthesized continuously at low levels c) They are often self-regulated (1) High concentrations of the regulatory protein binds to its own operator and represses RNA polymerase from binding to the promoter sequence (a) This stops transcription until its level drops III.THE LACTOSE OPERON A. Types of regulation 1. The lactose operon is under both positive and negative (inducible) regulation a) Inducible negative regulation (1) Negative (a) When repressor binds to operator, transcription does not occur (2) Inducible (a) When a derivative of the catabolic substrate lactose is present, it acts as an inducer (b) An inducer binds to the repressor (currently active – bound to the operator) and changes it to an inactive state (can no longer bind to operator) b) Positive regulation B. C. D. E. F. (1) CRP-cAMP molecule must bind to the operator to attract RNA polymerase biding (2) This will be discussed below in the section "Catabolic Repression" The lactose operon 1. Contains a promoter, operator, and three genes a) The lacY gene encodes -galactosidase permease protein b) The lacZ gene encodes -galactosidase c) The lacA gene? Overview of negative regulation 1. A bacterial cell does not want to waste the energy to synthesize enzymes to degrade an energy source if that energy source is not present a) The lactose operon is regulated to produce enzymes to break down lactose only if lactose is present 2. A lactose derivative acts as an inducer a) Lactose binds to the repressor molecule, converting it to an inactive form Mechanism of negative regulation 1. When lactose is absent a) The lac repressor protein is constituitively synthesized in its active form (1) The repressor binds the operator (2) This blocks RNA polymerase from binding to the promoter sequence (3) Genes for enzymes that degrade lactose will not be produced 2. When lactose is present a) Lactose acts as an inducer molecule and binds the lac repressor, thus deactivating it (1) The repressor falls off the operator (2) RNA polymerase can now bind promoter (3) Genes for enzymes that degrade lactose will now be produced Overview of positive regulation 1. If two sugars are present, glucose and lactose, Escherichia coli would prefer to use the glucose a) Therefore, the presence of glucose indirectly prevents the transcription of the lac operon Mechanism of positive regulation 1. IV.THE ARGININE OPERON A. Type of regulation 1. Repressible negative regulation a) Negative (1) When repressor binds to operator, transcription does not occur b) Repressible (1) When the anabolic product that is needed (arginine) is present, it acts as an corepressor (2) A corepressor binds to the repressor (currently inactive – not bound to the operator) and changes it to an active state (binds to operator) B. Overview 1. A bacterial cell does not want to waste the energy to synthesize enzymes to make an essential amino acid (arginine) if that molecule is present in the growth media a) The arginine operon is regulated to produce enzymes to make arginine only if that amino acid is absent (1) Enzymes for biosynthesis of arginine are highest when intracellular concentrations of arginine is low 2. Arginine acts as corepressor a) Arabinose binds to the repressor molecule, converting it to an active form C. Mechanism 1. Arabinose absent a) The Ara repressor protein is constituitively synthesized in its inactive form (1) The repressor will not bind to the operator (2) This means RNA polymerase will bind promoter and the genes for enzymes that synthesize arabinose will be produced 2. Arabinose present a) Arabinose acts as a corepressor molecule and binds the Ara repressor, thus activating it (1) The repressor binds to the operator blocking RNA polymerase from binding to the promoter region (2) Genes for enzymes that synthesize arginine will no longer be transcribed V. DIPTHERIA TOXIN GENE REGULATION A. Type of regulation 1. Repressible negative regulation a) Negative (1) When repressor binds to operator, transcription does not occur b) Repressible (1) When iron is absent, diphtheria toxin gene is turned on to kill host cells to obtain their iron (2) When the essential nutrient (iron) is present, it acts as an corepressor and binds to the repressor and changes it to an active state (binds to operator as to stop transcription) B. Overview 1. A bacterium (Corynebacterium diphtheriae) does not want to waste the energy to synthesize a toxin to kill host cells for the release its iron if iron is already present at sufficient concentrations a) The diphtheria toxin operon is regulated to produce toxin only if iron is absent 2. Iron acts as corepressor a) Iron binds to the repressor molecule, converting it to an active form C. Mechanism 1. In inactive repressor is constituitively synthesized 2. Iron absent a) The tox repressor protein is constituitively synthesized in its inactive form (1) The repressor will not bind to the operator (2) This means RNA polymerase can bind promoter and the genes for toxins to kill cells for their iron is produced 3. Iron present a) Iron acts as a corepressor molecule and binds the tox repressor, thus activating it (1) The repressor binds to the operator (2) RNA polymerase no longer bind to the promoter (3) Genes for diphtheria toxin will now be turned off VI.THE HISTIDINE OPERON A. Type of regulation 1. Attenuator regulation a) An attenuator is a stem loop structure found in RNA that can cause RNA polymerase to stop transcription (1) Stem loop forms from complimentary sequences on the mRNA, causing an area of double stranded RNA containing a single-stranded loop (2) This structure causes RNA polymerase to pause, and then carry out its exergonic nuclease activity, degrading RNA (instead of polymerization), till it no longer is bound to DNA (a) Thus transcription is stopped B. Overview 1. A bacterial cell does not want to waste the energy to synthesize enzymes to make an essential amino acid (histidine) if that amino acid is already present in the growth medium a) The histidine operon is regulated to produce enzymes to make histidine only if histidine is absent (1) Enzymes for the biosynthesis of histidine are at there highest level when the intracellular concentration of histidine is low 2. The absence of histidine causes the formation of the attenuator structure in the mRNA molecule a) Many stem loop structures can be formed by the leader sequence of the mRNA (1) There are six sequences that are complimentary to the complimentary sequence on both their sides (a) Sequence 1 can bind to sequence 2 (b) Sequence 2 can bind to sequence 1 or 3 (c) Sequence 3 can bind to sequence 2 or 4 (d) Sequence 4 can bind to sequence 3 or 5 (e) Sequence 5 can bind to sequence 4 or 6 (f) Sequence 6 can bind to sequence 5 b) One of these stem loops is an attenuator (1) The stem loop formed when regions 5 and 6 complimentary base pair is the attenuator (a) Prevents transcription from proceeding past the leader sequence (2) The attenuator stem loop forms when histidine is present (a) Hence enzymes for histidine synthesis will not occur C. Mechanism 1. Whether histidine is present or absent, RNA polymerase will initiate transcription a) Transcription of the entire operon will only occur if the attenuator structure does not form in the initially transcribe RNA (1) If the attenuator does form, transcription will be aborted after about 200 base pairs have been transcribed 2. The region upstream from the complimentary sequences contains many consecutive histidine codons a) If histidine is absent, the ribosome would stall here (1) The ribosome, stalled here, would prevent complimentary region one from being able to hybridize with complimentary region two b) If histidine is present, the ribosome will be directly behind the RNA polymerase (1) This means that as region 2 is synthesized, it would be able to bind to region 1 3. When histidine is absent the attenuator is not formed a) The ribosome stalls at the histidine codons near region 1 (1) 1 is prevented from binding to 2 (2) 2 binds to 3 (3) 4 binds to 5 (4) No attenuator (a) 6 cannot bind to 5, forming the attenuator, since 5 is with 4 4. When histidine is present the attenuator is formed a) The ribosome does not stall at the his codons near region 1, since histidine is present (1) Sequence 1 can bind to sequence 2 (2) Sequence 3 can bind to sequence 4 (3) Sequence 5 can bind to sequence 6 (a) This forms the attenuator stem loop (i) Caused RNA polymerase to fall off mRNA (b) This prevents the transcription of genes to make histidine VII.Escherichia coli OMP PROTEINS A. Type of regulation 1. Antisense regulation a) Antisense molecules are single-stranded RNA molecules that are complimentary to a portion of a specific mRNA molecule (1) If RNA molecules are complimentary, they will form double stranded RNA (a) Only single-stranded RNA can be translated, not double-stranded RNA (2) These are encoded by an antisense gene B. Overview 1. Escherichia coli can produce two different outer membrane proteins a) OmpF is produced under conditions of low osmotic pressure b) OmpC is produced under conditions of high osmotic pressure 2. If a pathogen changes molecules on its surface, it causes a delay in our immune responses, since we have to synthesize different antibodies 3. Escherichia coli changes its outer membrane porin proteins from one form to another, thus avoiding the host immune response C. Mechanism 1. Two regulatory genes encode antisense RNA molecules complimentary to two omp protein genes a) Only one of the antisense molecules is transcribed at a time b) Both omp protein genes are transcribed constituitively 2. Which ever regulatory gene being transcribed at a certain time will produce an antisense RNA molecule that will bind to one of the omp RNA molecules a) Therefore, this omp RNA molecule will not be translated, as it is now double stranded b) The other omp RNA molecule will be translated, as it has no antisense molecule at the time, and hence it is single stranded 3. When the regulatory gene being transcribed changes, the antisense molecule to the second omp protein gene will now be produced a) This will hybridize to the second omp RNA molecule, preventing its transcription b) This first omp RNA molecule will no longer be ties up by its antisense RNA and therefore will be free to be transcribed VIII.Salmonella FLAGELLIN GENE A. Overview 1. Humans make antibodies against specific molecules on infectious agents to help rid the pathogens from our bodies. 2. If a pathogen changes molecules on its surface, it causes a delay in our immune responses, since we have to synthesize different antibodies a) Salmonella can change the molecules in its flagella to avoid the host immune response B. Type of regulation and mechanism 1. Transposon mediated site specific inversion a) Transposons are DNA sequences which are removed from DNA and then reinserted (1) In this case, it removes itself, and reinserts itself in the same place, but in opposite orientation b) Two genes flank a promoter contained within a transposon (1) The orientation of the promoter determines which gene will be transcribed (2) This can be changed if the transposon inverts itself IX.GLOBAL REGULATION A. Sigma factors may be changed to regulate many genes at once 1. Causes a) Stress, aerobic / anaerobic changes, nutrient starvation, osmoticity, sporulation b) Global regulation negates the necessity of individually turning off one large set of genes and turning on a different set of genes 2. Mechanism a) RNA polymerase contains five subunits (1) 2, , ', and compose the holoenzyme (2) The first four subunits compose the core enzyme which is capable of catalyzing RNA synthesis (3) The sigma factor is responsible for binding to the promoter region of DNA b) By switching the type of sigma factor produced a different type of promoter will be recognized by the RNA polymerase (1) Hence, a whole different set of genes will be transcribed