http://www.unc.edu/courses/2009spring/envr/740/001 slide 1Begin 02/10/09In the case of lac, transcription cannot begin even when the polymerase has made a tight complex at the promoter. That is because a tetrameric product of a regulator gene, the lacI gene, is normally bound to the DNA, at the downstream edge of the promoter, blocking the start point. That region is shown in detail on the next overhead:[OH; binding of repressor at operator]The protein is called a repressor, and the site of binding is the operator. The operator is an example of a cis-acting control element, while the repressor protein Lac I is an example of a trans-acting product. In effect, then, the normal state of lac operon is that it is repressed. In fact, transcription is activated only in the presence of β-galactosides. The question of how activation is accomplished is answered in the following overhead, which is a cartoon of the control cycle ofthe lac operon:[OH; cartoon of control cycle of lac genes]The top panel shows normal situation with the lacI repressor occupying the operator. However, as the overhead indicates, the repressor contains a site at which a small molecule called an inducer can bind. In this example, the β-galactosides themselves serve as the inducers: the system works because when β-galactosides are in the cell environment, minimal amounts may enter the cell by diffusion through the plasma membrane without the help of the lacY product. When a β-galactoside binds to the repressor, the repressor changes conformation in the region of operator recognition sequence and the repressor loses its affinity for the operator and dissociates, leaving the polymerase free to initiate transcription. This mechanism of repressor regulation is called allosteric control. [OH; model structure of lac repressor]1In this cartoon, the repressor is represented in its normal tetrameric form, with two of the four units bound. This is actually an accurate representation – what is not shown, is that there are two additional weaker binding sites, 410 bp downstream or 83 bp upstream from the start point. Additional binding at one of the second sites forces the DNA into a loop and results in full repression. Interestingly, the polymerase binding to the repressor-bound DNA is enhanced, but aslong as the repressor is present, transcription cannot commence. That has the effect of “storing” the polymerase at the promoter site so that transcription can begin immediately in the presence ofan inducer. The next overhead shows the structure of a monomeric unit of the lac repressor. This is a composite, because there is not a structure of a complete repressor in the PDB. All structures in the PDB are truncated at either the oligomerization end or the DNA binding end. N-terminal isDNA binding domain, HTH motif, with hinge domain connecting to two core domains. Between the core domains is the binding site of the inducer. Finally, the C-terminal end contains a helix that serves to mediate the association of the monomeric proteins to a functioning tetramer.[OH; crystal structure of lac repressor]The next overhead shows a ribbon diagram generated from an actual crystal structure of the repressor, with no inducer and with an inducer analogue bound. Note color coded structural features: α-helices in red and β-sheets in yellow. The use of an inducer analogue, rather than a galactoside inducer is necessary in order to make a stable complex. The structure of the inducer analogue is shown on the next overhead:[OH; structure of IPTG; isopropylthioglucose]The compound is referred to by the acronym IPTG, which stands for “isopropylthioglucose”. TheDNA-binding domain is missing, so this is a version of the repressor “truncated” at the hinge. What is shown in these structures is the C-terminal helix by which the repressors associate as tetramers:[OH; repeat]2the inducer binding site and the hinge-point of attachment of the “headpiece”, which we don’t see but which contains the DNA binding motif at the N-terminus, where the protein has been truncated. The next overhead shows the repressor associated as a tetramer by the C-terminal helices, which illustrates how the association works.[OH; truncated repressors associated as tetramer]The points of truncation are shown on just two of the tetrameric units for clarity. The left hand panel shows the unbound repressor and the right panel shows the protein with the inducer analogues bound. The next overhead shows the same structure in the same orientation (as closelyas I could orient it) with two of the four units selected (i.e., two of the units have been removed by computer) to make the truncation points clearer.[OH; 2 units selected to illustrate truncation points] use cursor to draw loop of DNA.I was able to find a structure of a repressor dimer bound to the operator. This dimer is complexedwith “anti-inducers” – that is two ligands bound at the inducer site that freeze the repressor into aDNA-binding conformation.The anti-inducer is pictured as an inset – o-nitrophenylfructose, (acronym: ONPF). This structureillustrates the hinge and the DNA-binding region, which consists of two α-helices separated by a turn, which is a common DNA-binding motif called helix-turn-helix or referred to by the acronym HTH motif. (Remember the use of motif. In the figure, there is a small third helical segment just before the N-terminal.) The α-helices fit into the major groove of DNA, where they make contacts with specific bases. Binding of the inducer in the cleft has a very subtle effect on repressor core conformation, but it is likely that mechanism of inducer function causes the hinge to swing to an extent that the HTH DNA-binding region no longer associates snugly with the target sequence. The next overhead shows a comparison of monomeric units of the operator-bound repressor + anti-inducer with C-terminal truncation and the inducer-bound repressor with the N-terminal truncation. I have generated the slide with the repressors as closely as possible in 3the same orientation and as you can see, it is very hard to see any difference in the two core domains.An important and interesting question is how synthesis of the repressor is controlled? Ultimately,the cascade of control mechanisms cannot be infinite. In fact, there is no control over the lacI gene; however, initiation is relatively inefficient, so that there are never many excess repressor molecules present in the