BMB 462 Lecture 38 Outline of Last Lecture I. General principles of gene expressionII. Sigma FactorsIII. RNA promoter enhancersIV. Repression in the lac operonV. Sequence specific binding domainsOutline of Current Lecture I. Lac operon as an example of positive regulationII. Regulation of the trp operonIII. Attenuation in terminationIV. The SOS response to DNA damageV. Regulation at the Translation levelVI. Regulation of rRNA synthesisVII. Regulation of expression by small RNAsCurrent LectureConcepts to remembers from previous courses/lectures:- The lac operon experiences negative regulation via the lac repressor binding to the operator.I. Lac operon as an example of positive regulationa. The lac operon also provides an example for positive regulation.i. The CRP (cAMP Receptor Protein, aka CAP or Catabolite gene Activator Protein) binds to the CRP binding site at about -60 base pairs in the DNA.1. -60 is a typical site for activators to bind, since it is just upstream of where the RNA polymerase would bind, so it can help guide thepolymerase to the promoter.ii. CRP is a homodimer.iii. When cAMP binds to CRP, CRP binds to the palindromic CRP binding site and interacts directly with the α subunit of Sigma-70 RNA polymerase.These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.1. The core of the polymerase II is composed of α-2, β, β', and ω. Theα subunit in the polymerase is key for assembling the RNA polymerase, as well as interacting with activators of transcription.a. CRP is a good example of this secondary role; CRP performs activation by recruitment.2. At this point, it also bends DNA in such a way that puts strain on it so that it is easier for the polymerase to melt the bonds and separate strands for initiating transcription.a. Overall, CRP can provide a stimulation about 50-foldb. Maximum expression of the lac operon occurs when there is lactose present, but also when glucose is low (which means cAMP concentrations are high, and it can bind and activate CRP).i. If glucose is around, there's no need to initiate the lac operon. 1. There will still be a small amount of expression due to a lack of repression (due to the presence of lactose causing the repressor to fall off the operator sequence).c. CRP regulates many other genes besides the lac operon.i. CRP is a master regulator, meaning that it can regulate both positively andnegatively a whole regulon (a whole set of genes)1. Along with cAMP, its effector, CRP regulates the genes that encodeenzymes for metabolism of secondary sugars (sugars that are not glucose).a. This process is known as catabolite repression. This is somewhat of a misnomer, since CRP is an activator protein in this case. However, because glucose is the preferred carbon source, it is "repressing" the expression of things that could utilize other carbon sources.i. It is more accurate to view it as an absence of activation; when glucose is around, cAMP is low and CRP fails to activate the operons for genes that could metabolize many other sugars, such as the lac operon.d. In contrast to CRP, the lac repressor is a gene/operon specifc regulator.II. Regulation of the trp operona. The trp operon encodes for biosynthetic enzymesi. When there's no tryptophan - or any other of the 20 amino acids - available, E. coli has the ability to induce expression of enzymes that can synthesize whatever amino acid is needed.1. When tryptophan is available, however, the cell doesn't want to be expressing the trp operon.a. The first mechanism for shutting down the trp operon is the trp repressor. The repressor is a homodimer.i. The effector in this case is tryptophan; when it is present, it causes the repressor to bind to the trp operator sequence of the promoter region, thus preventing transcription.b. All DNA binding proteins have an affinity for the DNA, and while that affinity is high, it does not completely repress the operon.i. Therefor there is a second mechanism called attenuation.1. This regulation occurs at the transcript level, instead of regulating transcription initiation as the repressor does. Attenuation occurs after initiation of the transcript, and is responsible for deciding whether transcription should continue to the downstream genes.a. The attenuator is located in the leader region of the DNA and is a potential terminator of transcription, terminating transcription before the enzyme-encoding genes trpE, D, C,B, and A.c. The trp repressor has a helix-turn-helix motif that interacts with the palindromic operator.III. Attenuation in terminationa. Attenuation is transcription termination in the leader region of the DNA and depends on alternative RNA secondary structures regulated by availability of the charged Trp-tRNA to a translating ribosome.i. Attenuation depends on couple transcription and translation - since it has to do with whether the polymerase or not. This is what determines the alternate secondary structures in the leader region.1. Because of this, attenuation is not possible in eukaryotes (since transcription and translation are processes separated by location).b. The leader region of the mRNA contains four different sub-regions.i. The first region is the Leader peptide because it can be translated. There are 2 adjacent Trp codons in the leader peptide that play the crucial role in determining the secondary structures that decide if there's terminationof transcription.1. If Trp is present in the leader peptide sequence, termination occurs and now the downstream genes are transcribed.2. If Trp is absent, the protein forms a different secondary structure, termination does not occur, and the polymerase produces the long transcript.c. The site of transcription attenuation is located after the fourth sub-region and is characterized by a row of 'U's in the sequence.i. A series of 'U's is also key during ρ-independent mechanism of transcription termination. d. The entire leader region of the transcript is 162 nucleotides long. If transcription continues past this point, the next nucleotide begins the first gene of the biosynthetic enzymes, trpE.e. When tryptophan levels are high, the Trp-specific tRNA is going to be charged, since there is plenty of Trp around, and the cell wants to shut down the trp operon.i. As the trp gene is being transcribed, and the 2 Trp residues are present in the leader peptide, the ribosome is closely following the
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