prokaryotic gene regulation

• Understand the negative control of transcription involves types of proteins and important structural characteristics.
• Understand the positive control of transcription involves types of proteins and important structural characteristics.
• Understand how bacterial cells respond to heat stress involves the role the sigma-32 mRNA plays in detecting heat stress.
• Understand how gene expression is regulated at the lac operon involves understanding the structural characteristics of the lac operon and understanding the difference between a cis-acting and trans acting element.
• Understand how to predict the transcriptional state of the lac operon in partially diploid or haploid lac operon mutants involves understanding the structural characteristics of the lac operon and understanding the difference between a cis-acting and trans acting element.
• Understand how the trp operon is repressible involves understanding general negative feedback loops and why the cell would have them.
• Understand the concept of attenuation and how it works at the molecular level in the trp operon involves understanding the features of the trpL mRNA and their significance.
• Understand how mutations in various regions of the trp operon would affect the phenotype of a cell involves understanding the difference between constitutive transcription and regulated transcription.
• Codons 10 and 11 of the trpL region are tryptophan codons; with an adequate supply of tryptophan, these are easily translated, and the ribosome moves to the stop codon, partially obscuring regions 1 and 2.
• Mutating one of the two codons alters attenuator responsiveness to tryptophan, and mutating both of them abolishes the attenuator’s ability to sense tryptophan.
• If the tryptophan codons are altered to specify different amino acids, tryptophan production is affected by the level of that amino acid.
• This allows only regions 3 and 4 to pair, causing intrinsic termination of transcription by attenuation.
• Without an adequate supply of tryptophan, translation stalls at codons 10 and 11.
• The ribosome obscures region 1, allowing regions 2 and 3 to pair.
• This creates that antitermination conformation, allowing for continued transcription of genes needed for production of tryptophan by RNA polymerase.
• Mutation of regions 3 and 4, which prevents stable binding between them, also reduces the efficiency of the attenuation mechanism.
• Promoting transcription when trp is absent
• Lactose and Glucose present at a basal level lead to transcription of the lac operon.
• The binding of CAP-cAMP to the CAP binding site bends the DNA, opening up the promoter region, making it easier for RNA polymerase to bind.
• Mutations in the operator sequence (O C) are cis-acting, meaning they only influence the expression of the genes on the same chromosome.
• Hfr mapping revealed that the genes were very close together.
• Certain mutations of the lac operon lead to constitutive mutants, in which the genes are transcribed continuously whether or not lactose is available.
• Mutation in the lacI gene can generate a mutant repressor protein that cannot bind to the lacO site, leading to constitutive expression of the lac operon.
• The genotype of a partial diploid can be written as: F′ I + P + O + Z + Y − / I + P + O + Z − Y +.
• Constitutive mutations could affect the production of a regulatory protein (lacI) or its DNA-binding site (lacO).
• Other regulatory mutants cause cells to be unresponsive to the presence of lactose, and therefore lac− = noninducible.
• Glucose present leads to the production of only functional LacZ.
• In partial diploids, if lacI+ is also present, then the operon is inducible just like WT.
• The F′ copy of the operon is unable to produce a functional permease (lacY−) and the other copy is unable to produce functional β-galactosidase (lacZ−), but in combination, the mutations carried by each copy of the operon complement because the wild-type allele of each gene is dominant to the mutant allele.
• Mutational Analysis of the lac operon involved generating several dozen lac − mutants by treating E. coli with mutagens, assigning mutations into two complementation groups (lacZ and lacY genes), and carrying out complementation analysis in partial diploids produced by conjugation between F′ and F− bacteria.
• CAP binding to cAMP allows for DNA binding.
• The trp operon contains five structural genes and a regulatory region with a promoter (trpP), operator (trpO), and leader region (trpL) that contains the attenuator region.
• This suggests that a second mechanism regulates operon expression.
• A sixth gene outside the operon encodes the trpR protein, a repressor protein that is activated when bound to tryptophan.
• The trpL region contains four repeat DNA sequences and the mRNA produced contains complementary repeats as well as start and stop codons for a 14–amino acid polypeptide.
• Certain repressible operons have a second regulatory capability called attenuation, which can fine-tune transcription to match the immediate needs of the cell.
• trpR − strains have higher rates of operon transcription in the presence of tryptophan than trpR + strains but not 100%.
• trp operon expression attenuation is controlled by the 162-bp trpL region.
• The mutation in the lacI repressor causes the allosteric domain to be altered so that allolactose cannot bind to it, always bound to the operator, resulting in noninducible operon.
• The trp operon is a repressible operon involved in anabolic pathways that operate through activity of the end product to block transcription of the operon.