BMB400 PART FOUR - SUMMARYBMB400Part Four: Gene RegulationA SummaryThemes in mechanisms regulating the level of gene transcription1. Changes in chromatin structure can make the DNA template more accessible forassembly of the transcription preinitiation complex.a. Examples include the β-globin gene clusters in chickens and mammals. A locuscontrol region (LCR) is a distal regulatory sequence that is needed for opening ofthe chromatin domain and for high level expression of the genes. Both LCRs andsome enhancers can work by increasing the probability that a gene is in a permissiveenvironment for transcription (putative accessible chromatin).b. Much evidence has implicated enzymes such as histone acetyl transferases (HATs,e.g. protein complexes containing Gcn5p + Ada2 in yeast, PCAF + P300/CBP inmammals) and nucleosome remodeling ATPases (e.g. yeast SWI/SNF and itshomologs and analogs) in altering the chromatin so that the template is accessible.These are one type of co-activators of transcription, and can also be called adaptorsand mediators.c. Particular trans-activator proteins can be used to recruit activities such as HATs andremodeling enzymes to particular loci.d. One might expect chromatin-based mechanisms to be seen not only in eukaryoticorganisms but also in archaebacteria.2. Some negative regulatory proteins can block access of the transcriptional machinery topromoters.a. Binding of the yeast α2 repressor to its operator (binding site in DNA) will position anucleosome over the TATA box of the promoter, thus blocking access.b. An extensive complex of proteins is recruited to the silent mating-type loci in yeastand prevent expression of these genes by keeping them in an inaccessiblechromatin. Models postulate that these SIR proteins build a cage around thenucleosomal chromatin, keeping it inaccessible.c. The bacteriophage λ has a repressor and another negative regulator, called Cro, thatbind to specific sites (their operators) in the DNA. When bound to these sites, theyblock access of the RNA polymerase to the -10 and -35 boxes of the targetpromoters. The operator and promoter overlap in this case.3. Other negative regulatory proteins can still allow RNA polymerase to bind to thepromoter to form a closed complex, but they restrict the activity of the polymerase so that itdoes not initiate productive transcription.An example is the lac repressor bound to its operator. The operator does not overlapthe promoter, so both proteins can bind. However, the close proximity to therepressor prevents isomerization of the DNA-polymerase complex to the opencomplex.BMB400 PART FOUR - SUMMARY4. Some positive regulatory proteins (trans-activators) can increase the rate of transcriptioninitiation by directly contacting the RNA polymerase and recruiting it to bind more avidly tothe promoter.a. An example from eubacteria (E. coli) is the CAP protein, which in the presence ofcAMP will bind to a site centered at 62 bp upstream from the initiation site (-62) ofthe lac operon. When bound, the protein contacts the α subunits of RNApolymerase and increases the rate of transcription from the promoter. In otheroperons, cAMP-CAP binds to a site centered at -42 and again increases the rate oftransription. Note that the same regulatory protein (cAMP-CAP) can make differentcontacts with RNA polymerase at different operons and activate transcription bydifferent mechanisms, affecting the affinity of the polymerase for the promoter inone case and the rate of closed to open complex in the other.b. Another example is the repressor from bacteriophage λ, which infects E. coli. Itsoperator not only overlaps the promoter for the gene that it negatively regulates (seeabove), but it is adjacent to a promoter oriented in the opposite direction. It canstimulate transcription from this second promoter by direct contact with the RNApolymerase. The λ CII protein stimulates transcription from the otherwise weakpromoters PRE and Pint.c. In eukaryotes, there are several studies showing that the activation domains of sometrans-activators (e.g. GAL4-VP16) can bind to components of the basaltranscriptional machinery, in particular some of the TAFs that are associated withTBP in the general transcription factor TFIID. This may be a mechanism analogousto the CAP-RNA polymerase interaction for recruitment of the transcriptionalmachinery to the promoter. It may act in concert with the effects of trans-activatorsin establishing an open chromatin domain.5. Transcriptional regulators can change the conformation of the DNA, e.g. bending it, andthus allowing activation or repression. Examples include the action of cAMP-CAP at aMalT protein-DNA complex and HMG(I)Y in mammals.6. Although regulation of transcription initiation is a primary level of control in manygenetic systems, all subsequent steps in the pathway to gene expression are subject toregulation.a. Examples of regulation at transcription elongation include the trp operon in E. coli, inwhich the extent of translation of a leader peptide determines whether or not a ρ-independent terminator of transcription is used. An example from mammals is theHIV virus, in which a Tat protein acting at a TAR element close to the 5' end of themRNA will determine the efficiency of elongation past this sequence.b. Anti-termination at ρ-dependent sites in λ regulates the expresion of genes whoseproducts allow progress through the lytic cycle. [pp. 498-508; problem 4.16.]c. Examples of regulation of splicing abound in any system with alternative splicing.Particular splice enhancers and the proteins that interact with these segments ofexonic RNA are intensively studied in transcripts of sex-determination genes inDrosophila. [Part Three of the course.]d. Further regulation can be achieved during export of RNA from the nucleus,translation, post-translational modification, and degradation of both the mRNA andthe polypeptide. These are important steps, but beyond the scope of this