Molecular Biology RNDr. Jan Hošek, Ph.D. hosekj@vfu.cz TRANSCRIPTION POSTTRANSCRIPTION PROCESSING TRANSLATION POSTTRANSLATION PROCESSING REPLICATION  single type of RNA polymerase  nevertheless genes are take-on and take-off according to needs  prokaryotic cell must react to quick changes of environmental conditions  signals for starting and stopping transcription are in the form of small molecules - substrates  transport of signals to promoters are mediated by protein molecules Regulator - any substance, which participate on regulation of molecular process Regulatory protein - innate entity, which participate on the regulation - it usually binds to promoter Allosteric effector - substance of low molecular mass which by binding to regulatory protein change its conformation and by this its affinity to regulatory region Positive regulator - induces transcription, translation, … Negative regulator - inhibite transcription, translation, … change of regulatory protein conformation = interaction of an effector with regulatory protein extinction or creation of binding site for regulatory region change of regulatory protein conformation = interaction of an effector with regulatory protein extinction or creation of binding site for regulatory region RP RP E Negative allosteric effector = inhibites regulatory protein binding to regulatory region Positive allosteric effector = facilitates regulatory protein binding to regulatory region Negative regulatory proteins = their binding to regulatory region inhibite transcription of transcription unit by RNA polymerase Positive regulatory proteins = their binding to regulatory region enable transcription of transcription unit by RNA polymerase = activator of transcription regulator positive negative transcription START regulatory proteins enable binding regulatory protein to regulatory region molecular effectors bind to regulatory region bind to regulatory proteins pozitive negativepozitivenegative Inhibite binding regulatory protein to regulatory region transcription START transcription STOP transcription STOP NEGATIVE  it is a keystone of enzymatic induction and repression  binding of active repressor to operator stops transcription POZITIVE  it is a keystone of catabolic repression  binding of CAP to promoter in the presence of inductor stimulates transcription  Mostly for synthesis of enzymes which is stimulated by inducer Inducible enzymes Constitutive enzymes  their synthesis do not depend on the presence of inductor  they are produced in cell in constant amount transcription of genes is ON – inducible enzymes are produced inducer – repressor inactivation inactive complex promoter operator genes terminator represorrepresor transkription induktor  Mostly for the enzymes of biosynthetic pathways  The synthesis of these enzymes is inhibited by a specific metabolite of suitable metabolic pathway which is accumulated to critical amount and stops next synthesis  The synthesis is renewed if concentration of the metabolite fall down under the critical amount represor transcription of gene is OFF – synthesis of corepressor is stopped promoter operator genes terminator corepressor – activates repressor repressor http://www.mun.ca/biology/scarr/ bio4241_chapter13.htm  Substrate inhibits synthesis of inducible enzymes although inducer is still present  For example, glucose inhibits synthesis of β-galactosidase even in the presence of lactose as an inducer promoter operator genes terminator repressor inducer inactivates repressor inactive RNA polymerase has low affinity to the promoter, it is not binding, transcription is OFF or very slow Affinity of RNA polymerase is going up, it binds to promoter, transcription is ON complex CAP~cAMP – escalate affinity of RNA polymerase to promoter promoter operator genes terminator transcription   CAP inactive repressor CAP OCH2P OO OH O N N N N NH2 OH OCH2 OP O O O N N N N NH2 OH OCH2 OH H PO O O POH OH O P OO OH O N N N N NH2 OH adenylatcyclase phosphodiesterase GLC binding of RNA polymerase to promoter I: codes repressor Z: codes enzyme β-galaktosidase (lac  glu + gal) Y: codes enzyme permease A: codes enzyme thiogalaktosidtransacetylase Inducer = lactose the enzymes for metabolism of lactose a) glc is not present, lac is not present   repressor binds to operator promoter operator CAP repressor promoter operator CAP b) glc is not present, lac is present  enzyme induction c) glc is present, lac is present  catabolic repression represor promoter operator repressor represorrepressor for example trp operon contains genes necessary for tryptofan synthesis  The process of regulation of transcription based on the attenuator  Attenuator is a part of leader sequence of gene  Attenuator acts as an early terminator of transcription J. W. Roberts Science 328, 436-437 (2010) nonsense codon 1 XX 2 3 4 5 6 UUUUU protector (pause) preemptor (antiterminator) terminator XX – two codons for Trp promoter operator L genes terminator DNA mRNA 1 XX 2 3 4 5 61 XX 2 3 4 translation stops on the nonsense codon terminator 5 9 RNA polymerase releases here TRANSCRIPTION IS STOPPED 1 XX 2 Translation stops on the codons for Trp because this amino acid is in low number of copies preemptor TRANSCRIPTION FOLLOWS 1 XX 2 3 4 5 63 4 terminator is not formed https://www.youtube.com/watch?v=CeE83RyQFRo http://www.discoveryandinnovation.com/BIOL202/notes/lecture17.html AlexanderV.Yakhninetal. PNAS2008;105:16131-16136 • Secondary mRNA structure capable to bind molecular effector (ligand) • Binding of ligand on mRNA changes its secondary structure  the gene expression is „ON“ or „OFF“ https://sites.lsa.umich.edu/walter-lab/research/riboswitch/ Marie-Pier Caron et al. PNAS 2012;109:20182-20183 ©2012 by National Academy of Sciences http://www.yalescientific.org/2013/04/a-genetic-light-switch-riboswitches-shed-light-on-the-history-of-life/  Proteins which are induced by higher temperature  They are expressed by higher speed in the temperatures 42-45°C  Regulated by sigma factor sigma-32, which binds to other promoters than the standard sigma factor  Auto regulation by feedback loop = after accumulation the induced protein binds to sigma factor and releases it from RNA polymerase  Speedy of cell division is connected to speedy of ribosome formation  Synthesis of rRNA and tRNA is TAKE OFF if level of amino acid pool is low, synthesis of mRNA continues at least minimum amount