Dr Moni Kumari 28/04/2020 B.Sc. 3rd and M.Sc. 3rd Semester Transcriptional regulation in prokaryotes Gene is of two types Housekeeping genes/ constitutive gene: transcribed continually OR transcribed at a relatively constant level o Constitutive genes that are required for the maintenance of basic cellular function for the existence of cell. o Example actin, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin, transcriptional factors, insulin Facultative gene: transcribed when needed/ when a cell receives a signal from its surroundings. o Expression is influenced by environmental factors. Gene expression is the process by which the heritable information in a gene, the sequence of DNA base pairs, is made into a functional gene product, such as protein or RNA. The basic idea is that DNA is transcribed into RNA, which is then translated into proteins. Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are repressed, or turned off. The process of turning genes on and off is known as gene regulation Transcriptional regulation is the means by which a cell regulates the conversion of DNA to RNA (transcription), thereby coordinating gene activity. The regulation of transcription is a vital process in all living organisms. Transcriptional regulation – controlling the rate of gene transcription for example by helping or hindering RNA polymerase binding to DNA Gene upregulation: activation, or promotion – increase the rate of gene transcription Gene down-regulation repression, or suppression – decrease the rate of gene transcription There are many classes of regulatory DNA binding sites known as enhancers, insulators and silencers collectively called response element Transcription factors (TFs) are regulatory proteins whose function is to activate (or more rarely, to inhibit) or in layman terms control the rate of transcription by binding to specific DNA sequences. Domains of TFs are DBD, SSD, TAD 1. DBD: recognizes double- or single-stranded DNA 2. SSD: allows signaling molecules to bind the transcription factor 3. TAD: The transactivation domain (TAD) is where other proteins (co-regulatory proteins) bind to the transcription factor TFs turn on and off-genes in order to make sure that they are expressed in the right cell at the right time and in the right amount throughout the life of the cell and the organism A transcriptional activator is a protein that increases gene transcription of a gene or set of genes. Most activators are DNA-binding proteins that bind to enhancers or promoter- proximal elements. A transcriptional repressor is a DNA or RNA-binding protein that inhibits the expression of one or more genes by binding to the operator or associated silencers. A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes into messenger RNA. An RNA-binding repressor binds to the mRNA and prevents translation of the mRNA into protein. This blocking of expression is called repression. WAYS OF WORKING TF work alone If the transcription rate is up-regulating then TFs with other proteins in a complex, by promoting (as an activator) If the transcription rate is down-regulating then TFs with other proteins call repressor and form a complex TF+ACTIVATOR/REPRESSOR COMPLEX RECRUIT RNA POLYMERASE Transcription factors are proteins possessing domains that bind to the DNA of promoter or enhancer regions of specific genes. They also possess a domain that interacts with RNA polymerase II or other transcription factors and consequently regulates the amount of messenger RNA (mRNA) produced by the gene. TFs have defined DNA-binding domains with up to 106-fold higher affinity for their target sequences than for the remainder of the DNA strand. Coactivators A Coactivators is a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of a gene or set of genes A protein that works with transcription factors and activators to increase the rate of gene transcription Corepressor: A Corepressor is a molecule that can bind to repressor and make it bind to the operator tightly, which decreases transcription TFs+repressor+corepressor decrease the rate of gene transcription LAC OPERON The lac operon is an operon, or group of genes with a single promoter (transcribed as a single mRNA). The genes in the operon encode proteins that allow the bacteria to use lactose as an energy source. The lactose operon (lac operon) is an operon required for the transport and metabolism of lactose in E. coli and many other enteric bacteria. The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose When lactose is present, the lac genes are expressed because allolactose binds to the Lac repressor protein and keeps it from binding to the lac operator. Allolactose is an isomer of lactose. RNA polymerase can then bind to the promoter and transcribe the lac genes. The lac operon of E. coli contains genes involved in lactose metabolism. It's expressed only when lactose is present and glucose is absent. Two regulators turn the operon "on" and "off" in response to lactose and glucose levels: the lac repressor and catabolite activator protein (CAP). The lac repressor acts as a lactose sensor. It normally blocks transcription of the operon, but stops acting as a repressor when lactose is present. The lac repressor senses lactose indirectly, through its isomer allolactose. Catabolite activator protein (CAP) acts as a glucose sensor. It activates transcription of the operon, but only when glucose levels are low. CAP senses glucose indirectly, through the "hunger signal" molecule cAMP. STRUCTURE OF LAC OPERON Glucose control is accomplished because a glucose breakdown product inhibits formation of the CAP-cAMP complex required for RNA polymerase to attach at the lac promoter site When lactose is not available, the lac repressor binds tightly to the operator, preventing transcription by RNA polymerase. However, when lactose is present, the lac repressor loses its ability to bind DNA. It floats off the operator, clearing the way for RNA polymerase to transcribe the operon Glucose present, lactose absent: No transcription of the lac operon occurs. That's because the lac repressor remains bound to the operator and prevents transcription by RNA polymerase. Also, cAMP levels are low because glucose levels are high, so CAP is inactive and cannot bind DNA. Glucose present, lactose present: Low-level transcription of the lac operon occurs. The lac repressor is released from the operator because the inducer (allolactose) is present. cAMP levels, however, are low because glucose is present. Thus, CAP remains inactive and cannot bind to DNA, so transcription only occurs at a low, leaky level. Glucose absent, lactose absent: Transcription of the lac operon occurs. cAMP levels are high because glucose levels are low, so CAP is active and will be bound to the DNA. However, the lac repressor will also be bound to the operator (due to the absence of allolactose), acting as a roadblock to RNA polymerase and preventing transcription. Glucose absent, lactose present: Strong transcription of the lac operon occurs. The lac repressor is released from the operator because the inducer (allolactose) is present. cAMP levels are high because glucose is absent, so CAP is active and bound to the DNA. CAP helps RNA polymerase bind to the promoter, permitting high levels of transcription. Summary of lac operon responses .