BIOLOGY 305 1st Edition Lecture 22 Outline of Last Lecture I. Introduction to Regulation via The Lac Operon II. Two Tools to Modify and Analyze OperonsIII. Analysis of MutantsIV. SummariesA. Lacl Repressor MutationsB. Phenotype Based on InducerC. How to solve lac operon questions given GenotypeD. Summary of the Lac Operon and its GenesV. Catabolite RepressionVI. DNAse I FootprintingOutline of Current Lecture I. The Tryptophan OperonII. AttenuationIII. Control of Other Biosynthetic PathwaysIV. Vocabulary and Sample QuestionsCurrent LectureI. The Tryptophan OperonThe tryptophan operon is an anabolic pathway, aka a pathway that works to assemble biomolecular molecules. Each gene contributes to an intermediate in a metabolic pathway as shown below. The end product (tryptophan) regulates the operon at the operator…How is it regulated?Anabolic paths are shut down when the end product is made in sufficient quantities.The repressor gene trpR is responsible to expressing a repressor protein. Tryptophan allostericallyaffects this repressor protein, affecting its ability to bind to the operator. Importantly, this repressor-Trp complex enables binding to the DNA operator and subsequent blocking of transcription. In the absence of tryptophan, RNA polymerase is able to bind to the promoter and enable transcription. Note that anabolic paths do not use inducers.Example: We use a northern blot to analyze expression levels of trpC as a result of mutants on trpR (which results in a mutated repressor). As expected, tryptophan presence decreases trpC expression. Because it was found that mutation of trpR made little difference in expression of trpC (only a little more expression when repressor gene is mutated), we can conclude that theremust be a second regulatory system. II. AttentuationAttentuation is the control of gene expression by premature termination transcription. Between the operator and the trpE gene there is a L and A gene. The L gene is a leader sequence which encodes for an RNA leader, which is translated into a Leader Peptide. A Leader Peptide has 14 amino-acids and includes two tryptophan. The A gene encodes for an attenuator, which is a longsequence of U’s.The RNA leader acts as a tryptophan detectorRecall: Hairpin loops in mRNA can results in termination of transcription as the RNA polymerase loses association with the DNA.Recall: Prokaryotes undergo simultaneous transcription and translation, thus the RNA leader is translated as it is transcribedThe resulting RNA leader from the L gene has two alternative conformations. The RNA has four regions between the two tryptophan codons and the attenuator. In high tryptophan levels, region 3 and 4 bind and terminate transcription. This is because this conformation of RNA leader resembles Rho-independent terminators. In low tryptophan levels, 2 and 3 bind and forma hairpin loop. The ribosome conducting translation becomes stuck while transcription proceeds. What are the effects of attenuation?Attenuation results in the fine-tuning of gene expression levels. With attenuation there is a more linear decrease in tryptophan biosynthesis as tryptophan levels increase. However without attenuation, the Trp biosynthesis rate greatly decreases at intermediate levels of tryptophan, having the effect of creating distinctive ‘levels’ of synthesis in accordance with existing product.III. Control of other Biosynthetic PathwaysThe Tryptophan Operon is certainly not the only type of amino-acid regulator:Type of Response to Drastic Environmental ChangeHeat-shock is the sudden exposure of a cell to high temperature. Proteins loose their conformation under high temperatures and degrade. Thus, heat shock results in the activation of enzymes that degrade denatured proteins. It also activates chaperons, which work to help proteins refold. Thus in response to drastic change in environment, gene expression must drastically change in responseSporulation is when a bacterium forms heat-resistant and desiccation-resistant (aka resistant to the loss of water) spores in response to great stressAlternative sigma-factorsRecall: In prokaryotes, the function of a sigma factor is to ensure that RNA polymerase binds to the DNA in a stable fashion only at the promoter and not at other sites and initiate transcriptionIt is then released once RNA polymerase bings, only one type of sigma factor usually usedThere are alternative sigma-factors, which bind at different sequences at the -10 and -30 regions:Either 70-type or 24-type sigma factors facilitate the binding of the core RNA enzyme to bind to the PrpoH gene and initiate transcription. This results in an mRNA product called rpoH, which translates into a new sigma factor called sigma-32. Sigma-32 associates with RNA polymerase that transcribe heat-shock genes. Thus gene expression profiles can be changed at global levels.IV. Vocabulary and Sample QuestionsInducers – small molecules in catabolic gene regulation that cause the repressor to dissociate form the operatorCo-repressor – the opposite of an inducer, in anabolic gene regulation, these molecules cause the repressor to bind to the operatoreffectors – inducers and co-repressors are both classes of theseattenuation – mediated by leader mRNA, attenuation is another level of regulation phage operons – similar to bacterial operons because they use the cellular machinery of the host bacterial cell to replicate, transcribe, and translate their genesalternate sigma factors – aid RNA polymerase in binding to promoters of heat-shock and sporulation genes so that more than a hundred genes located at different parts of the bacterial chromosome can be activated at the same time using a single protein factorSAMPLE QUESTIONS:1) In Trp operon, wild-type leader sequence contains two codons for Trp. Consier a mutant in which one of these two codons are deleted. Assume the repressor is non-functional. How much Trp is synthesized by this mutant in comparison to the wild-type when there is a moderate level of Trp present?2) What will be the level of expression of trp operon genes in double mutants lacking both the Trp repressor and the leader and attenuator sequences of the Trp operon? (with and without Trp present)3) What will be the expression level of trp operon gene in a mutant with a mismatch in the 3-4 terminator stem-loop? (Assume that the Trp repressor is not functional.)4) Below are the wild type trp operon leader sequence and a