Topic 3 Prokaryotic gene regulation.pdf

Foundation in Pharmacology PHRM2005 Dr. Ricky R Lareu Prokaryotic Gene Regulation 2 Key Concepts The Central Dogma in molecular biology Prokaryotic transcription and translation Regulator proteins – DNA binding proteins Activator and Repressor regulator proteins Monocistronic and polycistronic mRNA...

Foundation in Pharmacology PHRM2005 Dr. Ricky R Lareu Prokaryotic Gene Regulation 2 Key Concepts The Central Dogma in molecular biology Prokaryotic transcription and translation Regulator proteins – DNA binding proteins Activator and Repressor regulator proteins Monocistronic and polycistronic mRNA Operons & regulons See Lecture Outline document for Key Concepts, Learning Outcome and Lecture Summary Reading and reference material: Mims’ Medical Microbiology, Chapter 2 ▪ Gene Expression: pages 11-16 3 The Central Dogma in Molecular Biology Revision Describes the two-step process, transcription and translation, by which the information in genes flows into proteins: DNA → RNA → protein. DNA Transcription RNA Translation protein 4 Flow of Information is Regulated by Proteins Revision Replication DNA Transcription Regulation RNA Translation protein 5 Nucleic Acids: DNA and RNA Revision DNA/RNA = long chains of nucleotides joined by phosphodiester bonds Nucleotide = a nitrogenous base, a heterocyclic sugar (ribose or deoxyribose) and at least one phosphate group. There are four nitrogenous bases in DNA Guanine, Cytosine, Adenine and Thymine In RNA Uracil replaces Thymine in RNA National Institutes of Health National Human Genome Research Institute Division of Intramural Research 6 DNA structure - Chemical Revision DNA is a polynucleotide Inter-strand hydrogen bonding stabilises the structure Nitrogenous bases bond preferentially to form complementary pairs A:T and C:G Purines to pyrimidines 7 https://commons.wikimedia.org/wiki/File:DNA_chemical_structure.svg#/media/File:DNA_chemical_structure.svg DNA Structure – Double Helix Revision DNA forms a double stranded molecule Double helix – twisted ladder configuration A sugar–phosphate backbone on the outside of the helix Nitrogenous base pairs are stacked in the centre of the helix DNA is very stable 8 DNA structures: Chromosomes Revision Folding of 1 In eukaryotic cells genetic material exists as highly organised structures chromosomes Takes other forms in bacteria and viruses The DNA in a human cell is almost a metre long Requires highly efficient packaging Millions of base pairs in length Relative Folding of 8000 cf naked DNA 9 See table 35.2 Harpers How to Write DNA Sequences Revision 5’ - ATGCCGTCT - 3’ 3’ - TACGGCAGA - 5’ When we write DNA we write 5 prime to 3 prime 5’- ATGCCGTCT - 3’: this is known as the positive or coding strand The complementary strand or template strand is 3’ - TACGGCAGA - 5’ But when single stranded we write it in the reverse complement direction = 5’- AGACGGCAT - 3’ 10 Ribonucleic acid (RNA) Revision RNA is a single stranded molecule RNA is far less stable than DNA RNA molecules have many functions in the cell Messenger RNA carrying information Catalytic in ribosomes Transfer RNA Regulatory RNAs Some viruses have RNA genomes 11 Proteins Revision Proteins are the main functional unit of the cell are composed of chains of amino acids, 20 naturally occurring a.a. The peptide bond How do we write protein sequences =GlutamineLysineSerineValine =GlnLysSerVal =QKSV 12 Transcription Revision The general mechanism of transcription is the same for both eukaryotes and prokaryotes (bacteria). However, there are subtle differences in the type of RNA polymerase and a major difference in that there is no nuclear membrane separating transcription from translation in prokaryotes, therefore these two processes can occur simultaneously in prokaryotes. Watch the following video on RNA synthesis: https://www.youtube.com/watch?v=vLz2A1cjPH8 13 Translation Revision The general mechanism of translation is also the same for both eukaryotes and prokaryotes. However, there are subtle differences in the type of ribosome. Again, since there is no nuclear membrane, translation can proceed on the 5’end of the new and emerging mRNA. This coupled process enables rapid and efficient response to changes in the bacteria’s environment. Watch the following video on protein synthesis: https://www.youtube.com/watch?v=yUJzqAU0xdo End of Revision material 14 Basic Concepts in Gene Regulation The processes describing the ‘decoding’ of genetic information to produce a functional molecule (protein, RNA) Organisms must regulate gene expression: ▪ gene expression is tailored to the metabolic needs of the organism – bacteria are very efficient ▪ Bacteria can quickly switch to a new carbon source, produce certain amino acids when absent, turn on virulence proteins (e.g. toxins) to infect cells Constitutive Genes – on all the time, basic cellular processes Regulated Genes – turned ‘on’ or ‘off’ ▪ Inducible – can be turned ‘on’ or upregulated ▪ Repressible – can be turned ‘off’ or downregulated Genes are regulated by DNA binding proteins 15 Transcription in Prokaryotes DNA copied by DNA-dependent RNA polymerase (RNA pol) to produce mRNA RNA pol binds to a promoter region upstream of the gene, initiating polymerization of mRNA strand Amount mRNA produced depends on the frequency and strength of RNA pol attachment Frequency of attachment is affected by: sequence of DNA promotor region, level of DNA supercoiling, and other regulatory proteins that bind adjacent DNA regions called DNA binding proteins Rifampicin binds to and inhibits the action of RNA polymerase – higher affinity than for human equivalent 16 Translation in Prokaryotes The ribosome binds to a specific sequence on the mRNA, with an initiator tRNA (fmet*-tRNA) The start codon initiates translation and the peptide elongates as the ribosomal complex moves along the mRNA Chain termination happens at one of three stop codons Many ribosomes can bind to the mRNA and generate many proteins very quickly, called polysomes Remember that translation can start before transcription of the new mRNA strand is complete. Why? *formylmethionine www.thomas-schlitt.net 17 Translation in Prokaryotes cont. www.chegg.com Although the process between prokaryotic and eukaryotic cells is similar, it is complex and there is enough difference in proteins and enzymes to exploit with antimicrobial agents Example: Bacteria use a 70S ribosomal complex which is different from the eukaryotic 80S – target for aminoglycosides Other antimicrobials that inhibit areas of protein synthesis include macrolides (e.g. erythromycin), tetracyclines, chloramphenicol, lincosamides 18 Genes and Operons Bacterial genes are usually part of an operon: ▪ coordination of functionally related genes ▪ efficient packing of genes on chromosome One promoter to one gene ▪ Monocistronic mRNA ▪ Found in eukaryotes One promoter for multiple genes ▪ Polycistronic mRNA ▪ Found in prokaryotes Fig. 2.7 Mim’s MM 19 DNA Binding Proteins Operator site – DNA segment adjacent to or overlapping promotor site where specific regulatory proteins bind Can enhance or suppress transcription DNA binding proteins (BPs) are known as trans-acting factors – diffusible and can act distally BPs bind to regulatory DNA sequences (e.g. promotor, operator) – these segments are known as cis-acting elements – regulate genes to which they are located adjacently Trans-acting factors RNA pol BP mRNA P O Cis-acting elements gene 20 Regulator Proteins Alter Gene Expression Regulator proteins are DNA BPs that alter gene expression: ▪ Activator & Repressor proteins Activation – positive gene regulation Activator protein binds to Operator site to allow/enhance RNA pol binding → turn on/increase mRNA quantity No Activator → none/background mRNA Repression – negative gene regulation Repressor protein binds to Operator site and inhibits/reduces RNA pol binding → none/lower mRNA quant. No Repressor → mRNA Fig. 2.8 Mim’s MM 21 Regulon – Multiple Gene Regulation Regulons are sets of operons (inc. promotor-operator-genes complex) that are controlled by the one regulator protein This synchronises large and complex processes Example ▪ The CAP regulator controls over 100 genes in E. coli (see Lac Operon slides) Fig. 2.8 Mim’s MM 22 Lac Operon – Inducible Regulation Bacteria preferentially metabolise glucose and suppress the metabolism of other sugars e.g. lactose → catabolite repression However, if no glucose available and lactose is, e.g. from milk, the genes that transport and metabolise lactose are turned on ▪ Lactose Operon is an example of an inducible operon Transcribed as one polycistronic mRNA β-galactosidase: lactose → galactose + glucose permease: transports lactose into the cell transacetylase: uncertain function Lactose 23 Lac Operon – Inducible Regulation cont. Regulator is CAP (cAMP-dependent catabolite activator protein) For CAP to bind to Operator the cell needs cAMP cAMP levels are low when glucose is available & high when no glucose (A) When glucose is absent and lactose is present CAP+cAMP bind to operator→ RNA pol → ↑mRNA (B) When glucose is available there are low cAMP and CAP cannot bind to Operator → RNA pol cannot bind → no/↓ mRNA (A) (B) Fig. 2.9 Mim’s MM 24 Lac Operon – LacI Repression (C) Lac Operon is also subject to repression LacI is a repressor protein that is encoded just upstream of the Lac genes with its own promotor Fig. 2.9 Mim’s MM (C) In the absence of lactose & glucose, the LacI protein binds to the operator and inhibits RNA pol initiating transcription In scenarios (A) and (B) (in the previous slide) when lactose is present, the LacI protein is inhibited by an inducer protein: ▪ Allolactose – binding of allolactose to LacI protein causes a change which detaches LacI from operator/inhibits binding Therefore, Lac operon is an example of fine-tuning of gene regulation in bacteria to respond to environmental carbon sources Animation on the regulation of the Lac Operon https://www.youtube.com/watch?v=g9GvmofU6jQ 25 Reg. Virulence Genes – Diphtheria Toxin Virulence factors – components of bacteria that enable infection and cell-tissue damage e.g. toxins, pili Diphtheria toxin production by Corynebacterium diphtheriae ▪ Negative regulation by Reppressor protein DtxR if Fe present – Fe is essential for many cell processes ▪ In body fluids there is very little free Fe: sequestered by proteins (protective response) When Fe is present, Fe binds to DtxR → bind to operator and Fe inhibits toxin production RNA pol DtxR X P X Toxin O No mRNA During early infection, low levels of Fe in body fluids, DtxR cannot bind to operator → toxin production to damage cells DtxR and access intracellular Fe RNA pol P O X mRNA Toxin 26 Reg. Virulence Genes – Whooping Cough Two component system: virulence genes in Bordetella pertussis, whooping cough Sensor protein (BvgS) ▪ detects changes in environ e.g. temperature ▪ Activated by autophosphorylation Fig. 2.10 Mim’s MM → activates Activator protein (BvgA) (phosphor.) → BvgA binds to Operator of many genes (regulon) → pertussis toxin operon and other virulence-associated genes 27 Reg. Virulence Genes – P. aeruginosa Biofilm Quorum sensing – a mechanism by which certain genes are activated when a particular bacteria density is achieved Biofilm is produced by Pseudomonas aeruginosa during lung infections in cystic fibrosis patients ▪ Biofilms protect bacteria from the immune system and chemicals → difficult to treat When P. aeruginosa numbers reach a certain density, signalling compounds released into surroundings reach a certain threshold concentration Receptors are then activated and results in the activation of genes to produce a biofilm See next slide for figure 28 Reg. Virulence Genes – P. aeruginosa Biofilm cont. 29 Summary Bacteria use the processes of gene regulation to respond to environmental conditions, either increasing or decreasing the production of enzymes and proteins This process is very efficient in bacteria as transcription and translation are coupled and can occur simultaneously Bacteria possess operons which encode for more than one protein involved in the same regulatory pathway e.g. metabolism of lactose DNA binding proteins regulate gene expression by altering access of RNA polymerase to the promotor site: activation, repression the Lac Operon is a model of inducible regulation i.e. usually turned off except when lactose is present and glucose is absent Many virulence factors are regulated by binding proteins e.g. Diphtheria and Pertussis toxins, biofilm from P. aeruginosa 30 Summary of Gene Expression Regulation Gene Expression is Regulated at Different Levels in Bacteria Transcriptional Control Initiation of Transcription Translational Control Lifespan of mRNA Translation Rate Post-Translational Control Protein activation Feedback Inhibition 31