BMB 462 Lecture 37 Outline of Last Lecture I. Role of glycosylation in protein targetingII. Targeting for secretion, membrane integration, or degradationIII. Targeting to mitochondria and chloroplastsIV. Targeting to nucleusV. Basic principles of nuclear transportVI. Amino-terminal signal sequences for targetingVII. Protein degradationVIII. Ubiquitin and Proteasomes in degradationOutline of Current Lecture I. General principles of gene expressionII. Sigma FactorsIII. RNA promoter enhancersIV. Repression in the lac operonV. Sequence specific binding domainsCurrent LectureConcepts to remembers from previous courses/lectures:-I. General principles of gene expressiona. Genes are expressed at different levels, at different times in the cell cycle, and in different tissues.i. Every cell in our body has the same DNA but the cells differentiate in the body by expressing different genes.b. Genes can be regulated at many different points, but most regulation occurs during transcription because it is the most economical stopping point.i. The 7 processes during which regulation can occur:1. Transcription2. Post-transcriptional processingThese 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.a. mRNAs and protein-encoding genes are highly processed; there is alternative splicing and alternative polyadenylation.3. mRNA degradation (how quickly the transcript is turned over by RNases or miRNAs)4. Translation (miRNAs can regulate translation by inhibiting it for particular mRNAs)a. Regulation can also occur here by ribosomal proteins.5. Once the protein has been made, during post-translational processing or modifications, proteolytic processing, disulfide bondformation, etc.6. Protein degradation (affecting the half-life of a protein can regulate how much activity a protein has in a given cell).7. Protein targeting and transport.c. Even what we consider "house keeping genes", which need to always be expressed at a certain level, need to be regulated; this is used for growth control,etc.i. When a cell is capable of growing, it wants to make a lot of ribosomes, but if it is under starvation conditions, it wants to limit ribosome production.II. Sigma Factorsa. Sigma factors are the specificity factors in bacteria.i. They have consensus sequences in the -35 and -10 regions, which are recognized by sigma factor.1. Regulation here involves activators and repressors that modulate RNAP binding to the promoter region, or a subsequent step.a. It regulates the binding of the holoenzyme to the promoterby making extra interactions.i. Subsequent steps include initiation, or instances when the cell is subject to heat shock (regulated by level of Sigma-32), or steps dependent on activity (the anti-Sigma binds to the sigma factor to repress it).b. TBP is a specificity factor in eukaryotes that's analogous to sigma factors.i. TBP and TFIID assemble proteins at the promoter, and then finally polymerase II comes in to initiate transcription.III. RNA promoter enhancersa. RNA polymerase II promoters have enhancer sequences where activators or repressors would bind.b. Negative regulation - a bound repressor inhibits transcription.i. An effector, a small molecule, can either cause the repressor to fall off theDNA or bind to it.1. i.e. in the lac operon, allolactose is the effector and it causes the lac repressor to fall off the DNA so transcription can begin (negative regulation).2. i.e. the trp operon; if trp is around, you want to shut off the process, so the effector (trp) adds the trp repressor in binding to the operon (also negative regulation).ii. Positive regulation - bound activator facilitates transcription.1. In bacteria, the activator protein typically binds just upstream of where the polymerase is bound. (Repressors typically bind operator sequences that overlap the promoter, and often hinder the polymerase from binding).a. In eukaryotes, it is not so simple. i.e. Enhancers can be thousands of base pairs away but affecting the promoter through DNA looping.i. Enhancers are the sites where the sequence-specific DNA binding proteins, be they activators or repressors, bind in eukaryotic promoters.b. i.e. In the lac operon, the CRP (cAMP Repressor Protein) is the activator. It binds to cAMP, the effector.c. Master genes, or master regulators, control regulons (sets of genes) and many times, transcription factors act as activators of some genes and repressors of other genes.i. This would be simple, because the DNA could have a binding site in the promoter or in the transcription factor (in which case it acts as a repressor), or you could have a binding site just upstream of the gene (in which it acts as an activator).d. The cell could also have regulatory cascades, which are fairly common during developmental processes.i. One transcription factor turns on its regulon. In that regulon, there is one or more transcription factors that then activate their regulons, to create a growing affect of regulation in the cell.IV. Repression in the lac operona. The lacI gene encodes the lac repressor, which then binds to the operator O1 (of 3 operators in the vicinity of the lac operon)i. O1 is the main operator, so binding to it turns down expression ~100-fold.1. The O1 operator sequence is a palindrome that overlaps the +1 region, right where the polymerase needs to bind. Transcription factors are often palindromic.b. When lactose is available in the extracellular space, it can be transported in by galactoside permease (produced by lacY).i. There is always a little expression of the lac operon, even with repression,so that when there is lactose present in the environment, it can be transported into the cell.c. Then the dissacharide lactose is transglycosylated by lacZ in one pathway, which converts it to allolactose.i. Allolactose has a higher affinity to the lac operon than does lactose.ii. β-galactosidase (the main product of lacZ) is the main enzyme for utilization of lactose. It can cleave the disaccharide into the monosaccharides galactose and glucose, which can be fed into processes like glycolysis.d. The lac repressor binds as a tetramer to two operators (it is essentially a dimer ofdimers).i. Many transcription factors in bacteria are dimeric, while the lac repressor is tetrameric (4 monomers).ii. When it binds to 2 operators, you can get approximately 1000-fold repression by the lack repressor.e. When the Lac
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