BIOL 366 Lecture 11 on Transcriptional Regulation PDF

BIOL 366 Lecture 11 Transcriptional regulation of gene expression: Role of regulatory DNA-biding proteins 1) Text Section: 19.1 – 19.2; 20.1 (The SOS response) Key Terms: activation, repression, activator, repressor, regulatory site, positive regulation, negative regulation,...

BIOL 366 Lecture 11 Transcriptional regulation of gene expression: Role of regulatory DNA-biding proteins 1) Text Section: 19.1 – 19.2; 20.1 (The SOS response) Key Terms: activation, repression, activator, repressor, regulatory site, positive regulation, negative regulation, DNA looping, coactivator, corepressor, insulator, effector, polycistronic mRNA, operon, regulon. 1 Lectures 11 and 12 provide the background to zinc-finger nuclease, which will be discussed in lecture 12 in detail. 2 Promoter structure and gene expression in prokaryotic - Prokaryotic DNA is not highly condensed - Prokaryotic genes are mostly constitutively expressed - RNA Pol generally has access to every promoter - Promoters have a -10 and a -35 element -10 element, TATA box (aka Pribno box) has a consensus sequence: TATAAT/A -35 element usually consists of the six nucleotides TTGACA. - Regulatory molecules stimulate or inhibit RNA Pol binding to the promoter Operator: Promoter regions where repressors (of transcription) bind Activator binding site: Regulatory regions where activators (of transcription) bind 3  Gene expression: A few terms Polycistronic mRNA: A transcript (mRNA) that encodes several proteins Has a leader sequence preceding the first gene. Subsequent genes are followed by “intercistronic regions”. Operon: An “expression unit” consisting of one or more co-transcribed genes and the operator and promoter sequences that regulate their transcription. Regulon: – A group of genes or operons that are coordinately regulated. – Some, or all genes, may be spatially distant in the chromosome or genome. 4  Transcriptional regulation can be negative or positive. a) Negative regulation Repressor proteins are used to inhibit transcription A repressor-binding site overlaps the promoter. When the repressor protein binds, RNA polymerase cannot initiate transcription. Fig 19.2a 5  Transcriptional regulation can be negative or positive. b) Positive regulation: – Some genes have promoters that are unable to strongly bind to RNA Pol by themselves. – Promoters of these genes are controlled by activators An activator protein binds next to OR near the promoter The activator recruits RNA polymerase to the promoter Transcription is initiated Fig 19.2b 6  Transcriptional regulation from a distance: DNA looping. Some transcription activators bind a distant regulatory site, enabling the promoter / RNA Pol complex to form a DNA loop. Fig 19.3 7  DNA looping can be mediated by a single regulatory protein. Example: The bacterial Lac repressor - Is a protein, made of four identical subunits - It binds two distant sites on a single DNA molecule, forming a DNA loop. 8 Certain regulatory proteins play an architectural role. Some transcription regulators, known as architectural regulators, bend the DNA when they bind their DNA site, thus promoting looping. The regulator facilitates looping for recruitment of RNA polymerase by an upstream activator Example: Bacterial H-NS (histone-like nucleotide 9 structuring protein) Transcription coactivator. Is a protein Stimulates transcription by “bridging” the RNA Pol with the activator(s) Does not bind DNA directly. Example: The eukaryotic protein complex “Mediator”, which bridges RNA Pol II to proteins that bind distant regulatory regions on DNA. 10 Transcription corepressors. A protein that binds an activator to suppress transcription. Note: The corepressor does not bind RNA Pol Example: Yeast Cys8 protein. Regulates multiple genes. 11  Role of “Insulators” in control of gene expression DNA looping could lead to the activation of “non-target” genes Insulators are proteins that block the unintended effects of DNA looping Insulators are present in pro- and eukaryotes 12 Transcription “Insulators” ; an example CTCF (CCCTC-binding factor: - A highly conserved zinc finger protein Text Fig 21-60 13  The regulation of gene expression often involves effectors. Effector: - Is a small molecule (not protein) - Binds the activator or repressor of a given gene - Causes a conformational change in the activator / repressor protein - Results in an increase or decrease in transcription from the gene 14  Effectors can inactivate a repressor, enabling transcription. I. Repressor binds DNA in the absence of the effector. II. The effector binds the repressor causing its dissociation from DNA to permit transcription. 15  Effectors can also activate a repressor, shutting down transcription. I. The repressor (orange) is activated by binding the effector. II. The activated repressor- effector complex binds promoter, shutting down transcription. 16  Effectors can inactivate activators, turning transcription off. I. In the absence of the effector, activator binds DNA and RNA Pol and transcription proceeds II. When effector is present, it binds and inactivates the activator to inhibit transcription 17  Effectors can enable activators to stimulate transcription. I. In the absence of the effector, activator does not bind DNA and transcription cannot proceed II. When present, the effector binds and activates the “Activator (green)” III. The activator interacts with DNA and RNA Pol to stimulate transcription. 18  Global (coordinated) regulation of gene expression Refers to coordinated expression of several genes that may be near one another, or dispersed throughout the genome. Found in pro- and eukaryotes Most studied cases involve response to stress 19 Global regulation can occur by a common activator Activator may be expressed when needed A existing activator may become active By another protein By an effector FIGURE 19-12a: 20 Global regulation of groups of genes can also occur via removal of a common repressor, through: an effector proteolytic degradation of the repressor FIGURE 19-12b: 21  Coordinated Transcription of several genes: The SOS system. The expression of a number of genes involved in repair of damaged DNA in bacteria is induced in a coordinated manner. This is known as the SOS response. Genes involved in the SOS response are located at different sites in the chromosome. The SOS response requires two key regulatory proteins: – The RecA protein – LexA repressor protein 22  Coordinated Transcription of several genes: The SOS system. Fig 20-13a In the default (normal; no damage) state of the E. coli cell, the LexA repressor prevents transcription of the SOS genes. In response to DNA damage, LexA undergoes autocleavage, inactivating itself and allowing transcription of the SOS genes. 23  Coordinated Transcription of several genes: The SOS system Notes: Autocleavage of Fig 20-13b LexA repressor requires RecA protein. DNA damage creates sites of single-stranded DNA, which are quickly bound by RecA protein. DNA-bound RecA becomes a coprotease for LexA, and their association facilitates the destruction of LexA and induction of the SOS response. 24