transcription+initiation

· For reasons of economy, it makes most sense to regulate gene expression at the level of transcription initiation  · The active site of the RNA polymerase has determinants both for the DNA template and the RNA product. The active site is made of two subunits – β and β’. The ω subunit doesn’t appear to have any role in transcription, but it does act as a chaperone assisting the correct folding of the β’ subunit   · In RNA polymerase, the smaller carboxy-terminal domain of the α subunit (called α-CTD, found at residues 250-329) has an important role in the recognition of some promoters  · For RNA polymerase to bind to promoter, it must interact with σ factors to form a holoenzyme  · The σ factor has three functions:  o Recognition of promoter o Binding to promoter o Unwinding of DNA helix  · σ factors have common features, with the important exception of the σ54 family (this family obligately requires an activator for the RNA polymerase holoenzyme to access the open complex of the DNA) <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">σ factors are multi-domain (they may have up to 4 domains). Various domains are joined by linkers <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">Domains 2, 3 and 4 are involved in promoter recognition. The function of domain 1 is unknown, indeed it is often absent <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; margin-left: 72.0pt; margin-right: 0cm; margin-top: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level2 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> o <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">Promoter -10 (10bp upstream of the promoter) elements are recognised by domain 2 of the σ subunit <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; margin-left: 72.0pt; margin-right: 0cm; margin-top: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level2 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> o <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">Promoter -35 (35bp upstream of the promoter) elements are recognised by domain 4 of the σ subunit <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; margin-left: 72.0pt; margin-right: 0cm; margin-top: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level2 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> o <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">the extended -10 element is 3-4bp upstream of the -10 element, it is recognised by domain 3 of the σ subunit <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; margin-left: 72.0pt; margin-right: 0cm; margin-top: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">The ‘open complex’ is the unwinding of DNA around the transcription start site <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">The UP element is a ~20bp sequence upstream of the -35 element that is recognised by the C-terminal domain of RNA polymerase α subunit <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: 'Times New Roman',serif; font-size: 13pt;"> <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">The relative contribution of each promoter element to binding RNA polymerase varies between promoters <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">No promoter binds every element perfectly, or RNA polymerase would be bound too tightly - deficiencies in some elements are compensated by others <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">Isomerisation to create the transcription bubble (between -10 and +2) is poorly understood but results in domain 2 being bound to the non-template strand <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">RNA polymerase and σ factors are in short supply – promoters are in competition for the holoenzyme <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-add-space: auto; mso-layout-grid-align: none; mso-list: l2 level1 lfo2; mso-pagination: none; text-autospace: none; text-indent: -18.0pt;"> · <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;">Multiple mechanisms ensure ‘fair’ distribution of the holoenzyme between promoters. These mechanisms also regulate 'fine tuning' - i.e. up- or down-regulating sets of genes in response to environmental conditions. These mechanisms are described below. <span style="line-height: normal; margin-bottom: .0001pt; margin-bottom: 0cm; mso-layout-grid-align: none; mso-pagination: none; text-autospace: none;"><span style="font-family: "Times New Roman","serif"; font-size: 13.0pt;"> · ** <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Promoter sequence. **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> The presence of consensus-sequence promoter elements provides a greater affinity for RNA polymerase; many of the strongest bacterial promoters have effective UP elements, and these seem to function simply by binding to the RNA polymerase α-CTD. These so-called // cis // functions are static, however, and thus it is usually // trans //-acting factors which change in response to environmental stimuli <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> · ** <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Sigma factors. **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> // E. coli // has one main σ factor, σ70, which equips RNA polymerase to recognise most promoters. Some σ factors accumulate in response to environmental stresses and compete with σ70 for RNA polymerase. The heat-shock response, for example, causes accumulation of σH and σE, released in the cytoplasm and periplasm respectively, to induce the transcription of genes needed to cope in elevated temperatures. The activity of σ factors can be regulated by anti- σ factors which sequester them away from RNA polymerase, and in some cases proteolytically degrade them. The genomes of organisms who inhabit a wider diversity of environments seem to encode a corresponding wider variety of σ factors. Although it was initially believed that relative affinity played a role in σ-RNA polymerase binding, studies by Jishage et. al have indicated that small molecules may have a role in σ factor exchange. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> · ** <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Small ligands. **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> An example is guanosine 3’5’ bisphosphate (ppGpp) which is synthesised when amino acid availability is restricted causing translation to be restricted; it binds to RNA polymerase and destabilises open complexes to prevent synthesis of the translational machinery. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> · ** <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Transcription factors. **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> DNA-binding proteins that up- or down-regulate transcription. Seven TFs in // E. coli // are responsible for regulating 50% of all regulated genes (FNR, IHF, Fis, ArcA, NarL and Lrp). The DNA-binding domains can be regulated by small molecules whose concentration fluctuates with environmental conditions (e.g. allolactose in the lac operon). Covalent modifications can also be used to regulate transcription factors, as in the case of NarL, which can only bind to DNA when phosphorylated by sensor kinase (which itself is activated by binding to nitrite ions). Other modes of regulation are controlling synthesis and degradation, and the sequestering of transcription factors by regulatory proteins. Some transcription factors act solely as activators or repressors; others can act as either depending on the promoter to which they bind. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Activators can increase RNA polymerase affinity by // simple activation // of which there are 3 mechanisms: <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">1. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Class I activation, where the activator binds upstream of the -35 element and increases RNA polymerase affinity by binding to the αCTD. <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">2. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Class II activation, where the activator binds to an overlapping region of the -35 element and binds to domain 4 of the RNA polymerase σ subunit <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">3. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Class III activation, where the conformation of the promoter is altered to increase the affinity of RNA polymerase with the -10 and/or -35 elements <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Repressors restrict the access of RNA polymerase for the promoter via // simple repression // of which there are also 3 mechanisms: <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">1. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Steric hindrance of RNA polymerase <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">2. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Interference with the post-recruitment steps of RNA polymerase <span style="font-family: 'Times New Roman',serif; font-size: 13pt; line-height: 115%;">3. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Multiple repressors binding to promoter-distal sites to cause repression by DNA looping <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Many transcription factors cannot be classified, or their function is unknown. <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> · ** <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Chromosome folding. **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> Nucleoid proteins which alter the accessibility of chromosomal DNA fluctuate in concentration in response to environmental stimuli. We are ignorant as to whether there is a bacterial chromatin structural unit, so general rules for this process have not been identified <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;"> <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">
 * <span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">Power of Bacterial Genomics – Notes on Transcription Initiation document **<span style="font-family: "Times New Roman","serif"; font-size: 13.0pt; line-height: 115%;">