Lecture 7.doc

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University of Alberta
Microbiology (Biological Sciences)
Nicolas Vozza

Lecture 7 1 Transcriptional Regulation Because gene expression is metabolically expensive. Control of gene expression is essential to LIFE Genes in prokaryotes regulated together transcriptionally are called operons. Co-regulation of genes used for a given situation. Genes must be regulated so that energy is used efficiently. Late 1950’s, the 1 example of a regulatory protein was discovered during a genetic study of the E. coli lac operon. Monod et al. noted mutants that showed constitutive expression of β-galactosidase rather than inducible, normal expression. Induction could be restored by introducing genetic material in trans. Postulated that the DNA encoded a repressor (lacI). This led to the notion that ALL regulation was due to repressors. Not until the late 1960’s did experiments with arabinose and maltose operons challenge this view. Lecture 7 2 Mutations in these pathways led to non-inducibility Induction was restored by in trans DNA. Now, hundreds of activators and repressors are known. Some are specific acting at a few loci (lacI) Others are “global” regulators controlling 100’s of genes (fur) Sequence analysis shows that most activators an repressors belong to a small number of families . Lecture 7 3 Both genetics and biochemistry have been used to identify binding sites for transcription factors. Base substitutions > binding interference and footprinting. Sequence analysis and in silico methods nowadays. In most cases, activator binding sites are located upstream of repressor binding sites. For σ70, activators bind –30 to –100 For repressors, they overlap the RNAP binding site. -35, -10. Physically stop the RNAP from binding. 4 functions of regulators 1. Bind specific DNA sequences. 2. Multimerize for increased stability on DNA. 3. Directly contact and recruit RNAP. 4. Their activity can be modief (by other proteins). It turns out that most Tn factors are made up of modules, & these modules appear in a number of different proteins. Lecture 7 4 Domains 1. DNA-binding domains – A. Helix turn helix (HTH) B. Zinc fingers (rare in prokaryotes) C. Leucine zipper (rare) 2. Oligomerization – Dimers or tetramers (increases the concentration of activating signals on DNA) 3. Activation domains – (interact with RNAP through acidic amino acids). Activator or Repressor? Are there rules to determine whether a regulator functions Lecture 7 5 as a repressor or activator? Site of Binding – Activators Prefer -30 or -90. Repressors Are downstream of -30. No Activators are known to bind downstream of -30 (would interfere with RNAP binding). SIMPLE ACTIVATION Single Tn activator stimulates Tn initiation. Can be studied easily in vitro 1. E. coli lac promoter+ CRP (cyclic AMP recognition protein). 2. λ CI repressor and P RMpromoter 3. MerR activator of merT (Involved in resistance to mercury). These studies show that activators accelerate the formation of open promoter complexes that are similar to those that form at activator-independent promoters. Lecture 7 6 Because of the competition for RNAP in the cell, small differences in promoter efficiency will result in big dif
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