Rna Polymerase First Bound To Operator Or Promoter

"half-site"

"half-site"

tac operator

FIGURE 16-7 The symmetric half-sites of the lac operator.

CAP-binding site DMA covered by RNA polymerase j —— » mRNA

3\ IsTTGCWT^re&CA^TCAftTCGASTgftGTAATCCGTQtJGar^

DNA covered by repressor

FIGURE 16-8 The control region of the lac operon. The nudeotide sequence and organization of the lac operon control region are shown. The colored bars above and below the DNA show regions covered by RNA polymerase and the reguiaioty proteins Note that Uc repressor ewers more DNA than that sequence defined as the minimal operator binding site, and RNA polymerase more than that defined by ihe sequences that make up the promoter

The lac operator overlaps the promoter, and so repressor bound to the operator physically prevents RNA polymerase from binding to the promoter and thus initiating RNA synthesis (see Figure JO-8). Protein binding sites in DNA can be identified, and their location mapped, using DNA foot printing and gel mobility assays described in Box 16-1. Detecting DNA-Binding Sites.

Box 16-1 Detecting DNA-Binding Sites

DNA Footprinting

How can 8 protein binding site in DNA, such as an operator, be identified? A series of powerful approaches allows identification of the sites where proteins act and the chemical groups in DMA (methyl, amino, or phosphate) a protein contacts. The basic principle that underlies these methods, is as follows. If a DNA fragment is labeled with a radioactive atom only at one end of one strand, the ¡oration of any break in this strand can be deduced from the size of the labeled fragment that results. The size, in turn, can be determined by high-resolution electrophoresis in a potyacrylamide gel. In the nuclease protection footprinting method, the binding site is marked by intemudeotide bonds that are shielded from the cutting action of a nudease by the binding protein (Box 16-1 Figure 1) The resulting "ftx>tpnnt" is revealed by the absence of bands of particular sizes The related chemical protection footprinting method relies on the ability of a bound protein to protect bases in the binding site from base-specific chemical reagents that {after a further reaction) give rise to backbone cuts.

By changing the order of the first two steps, a third method, chemical interference footprinting, determines which features of the DNA structure are necessary for the protein to bind. An average of one chemical change per DNA is made, and then the modified DNA is mixed with the binding protein. Protein-DNA complexes aie isolated, if a modification at a particular site does not prevent binding of the protein, DNA isolated from the complex will contain that modification and the harmless modification allows the DNA to be broken at (his site by further chemical treatment. If, on the other hand, a modification blocks DNA binding, then no DNA modified at the site will be found completed with btnding protein and the isolated fragments will not be broken at this site by subsequent chemical treatment. By using all three methods, we can learn where a protein makes specific contacts both with bases and with the phosphates in the sugar-phosphate backbone of DNA.

Gel Mobility Shift Assay

As just noted, how far a DNA molecule migrates during gel electrophoresis vanes with size: the smaller the molecule the more easily it moves through the get, and so the further it gets tn a given time. In addition, if a given DNA molecule lias a protein bound to it, migration of that DNA protein complex through the gel is retardixl compared to migration of the unbound DNA molecule. This forms the basis of an assay to detect specific DNA btnding activities. The general approach is as follows. A short DNA fragment containing the binding site of interest js tadioactively iabeltxi so it can be detected in small quantities by polyacrylamide gel electrophoresis and autoradiography. Ihis DNA "probe" is then mixed with the protein of interest and the mixture is run on a gel. If the protein binds to the probe, a band appears higher up the gel than bands formed from free DNA (see Box 16-1 Figure 2).

This method can be used to identify multiple proteins in a crude extract. Thus, if that probe has sites for a number of proteins found in a given ceil type, and that probe is mixed with an extract of that cell type, multiple bands can often be resolved. This ¡5 because proteins of different size will affect migration of the DNA fragment to different extents—the larger the protein the slower the migration. In this way, for example, the various transcriptional regulators that bind to the regulatory-region of a given gene can be identified.

BOX 16-1 FIGURE 1 footpfirning method. The stars represent the radioactive labels at the ends of the DNA fragments, arrows indicate sites where DNAse cuts, and red circles represent Lac repressor bound to operator. On the left, DNA molecules cut at random by DNAse are separated by size by gel electrophoresis. On the right, DMA mote-cutes are first bound to repressor thai subjected to DNAse treatment. The "footprint" is indicated on the right. Tbts corresponds to the collection of fragments generated by DNAse cutting at sites tn free DMA, but not in DMA with repressor bound to it In the latter case, those sites are inaccessible because they are within the operator sequence and hence cov ered by repressor.

DNA fragment DNA fragment +

DNA-b<nding protein

3 free ONA

bound DNA

free DNA

3 free ONA

bound DNA

free DNA

BOX 16-1 FIGURE 2 Gel mobility shift assay. The principle of the mobility shift assay is shown schematically, A protein is mixed with radiolabeled probe DNA containing a binding site for that protein. Tne mixture is resold by actyiamide gei electrophoresis and visualized using autoradiography. DMA not mixed wth protein runs as a single band corresponding to the size of the DNA fragment (left lane). In the mixture with the protein, a proportion of the DMA moiecuJes (but not alt of them at the concentrations used) binds the DMA molecule, thus, in the right-hand lane, there is a band corresponding to tree DMA, and another corresponding to the DNA fragment in complex with the protein.

FIGURE 16-9 Activation at the lac promoter by CAP. RNA polymerase binding at the he promoter with thu help o( CAP. CAP is recognized by the CTDs of the a subunits. The qCTDs also contact DMA, adjacent to the CAP site, when interacting with CAR As in Chapter 12, we use this representation of RNA polymerase when indicating specific points of con-tad between an activator and its target site on polymerase, or between regions of polymerase and the promoter.

As wf; have seen, RNA polymerase bintls the lac promoter poorly in the absence of CAR, even when there is no active repressor present. This is because the sequence of the - 35 region of the lac promoter is not optimal for its binding, and the promoter lacks an UP-element (see Chapter 12 and Figure 16-0). This is typical of promoters that are controlled by activators.

CAP binds as a dimer to a site similar in length to the lac operator, but different in sequence, This site is located some 60 bp upstream of the start site of transcription [see Figure 16-8), When CAP binds to that site, the activator helps polymerase bind to the promoter by interacting with the enzyme and recruiting it to the promoter (see Figure 16-6). Tliis cooperative binding stabilizes the binding of polymerase to the promoter. We now look at CAP-mediated activation in more detail,

CAP Has Separate Activating and DN A-Binding Surfaces

Various experiments support the view that CAP activates the lac genes by simple recruitment of RNA polymerase. Mutant versions of CAP have been isolated that bind DNA but do not activate transcription. The existence of these so-called positive control mutants demonstrates that, to activate transcription, the activator must do more than simply bind DNA near the promoter. Thus, activation is not caused by, for example, the activator changing local DNA structure. The amino acid substitutions in the positive control mutants identify the region of CAP that touches polymerase, called the activating region.

Where does the activating region of CAP touch RNA polymerase? when activating the lac genes? This site is revealed by mutant forms of polymerase that can transcribe most genes normally, but cannot be activated by CAP at the lac genes. These mutants have amino acid substitutions in the C-terminal domain (CTD) of die a subunit of RNA polymerase. As vre sari' in Chapter 12, this domain is attached to the N-terminal domain (NTD) of a by a flexible linker. The aNTD is embedded in the body of the enzyme, but the urCTD extends out from it and binds the UP-element of the promoter (when that element is present) (see Figure 12-7).

At the lac promoter, where there is no UP-element, uCTD binds to CAP and adjacent DNA instead (Figure 16-9). This picture is supported by a crystal structure of a complex containing CAP, uCTD, and a DNA oligonucleotide duplex containing a CAP site end an adjacent UP-element (Figure 16-10). In Box 16-2, Activator Bypass Experiments. we describe an experiment showing that activation of the lac promoter requires no more than polymerase recruitment.

Haying seen how CAP activates transcription at the lac operon — and how Lac repressor counters that effect—wc now look more closely at how these regulators recognize their DNA-binding sites.

FIGURE 16-10 Structure of CAP-«CTD-DNA complex CAP ts shown bound as a dimer to its site just as we saw in Figure 5-18. In addition, in this case, the «CIO of RNA polymerase is shown bound to an adjacent stretch of DMA, and interacting with CAP. The Sife of interaction on each protein involves the residues identified genetically. In this figure, CAP «s shown in turquoise and the «CTD <jf pofymerase in purple. One molecule of ATP is shown bound to each monomer of CAP. (Bcnoff B. et si 2002. Science 297: 1562.) Image prepared with MolScript and Raster 3D.

FIGURE 16-10 Structure of CAP-«CTD-DNA complex CAP ts shown bound as a dimer to its site just as we saw in Figure 5-18. In addition, in this case, the «CIO of RNA polymerase is shown bound to an adjacent stretch of DMA, and interacting with CAP. The Sife of interaction on each protein involves the residues identified genetically. In this figure, CAP «s shown in turquoise and the «CTD <jf pofymerase in purple. One molecule of ATP is shown bound to each monomer of CAP. (Bcnoff B. et si 2002. Science 297: 1562.) Image prepared with MolScript and Raster 3D.

CAP and Lac Repressor Bind DNA Using a Common Structural Motif

X-ray crystallography has boon used to determine the structural basis of DNA binding for a number of bacterial activators and repressors, including CAP and the Lac repressor. Although the details differ, the basic mechanism of DNA recognition is similar for most bacterial regulators, as we now describe.

In the typical case, the protein binds as a homodimer to a site that is an inverted repeat (or near repeat). One monomer binds each half-site, with the axis of symmetry of the dimer lying over that of the binding site (as we ssw for Lac repressor, Figure 16-7). Recognition of specific DNA sequences is achieved using a conserved region of secondary structure called a helix-turn-helix (Figure 16-11). This domain is composed of two a helices, one of which—the recognition helix—fits into the major groove of the DNA. As we discussed in Chapter 5, anu helix is just the right size to fit into the major groove, allowing amino acid

Box 16-2 Activator Bypass Experiments

If en activator has only to recruit polymerase to the gene, then other ways of bringing the polymerase to the gene should work just as well. This turns out to be true of the lac genes, as shown by the following experiments (6ox 16-2 Figure !).

In one experiment, another protein:protein interaction is used in place of that between CAP and polymerase. This ts done by taking two proteins known to interact with each other, attaching one id a DMA-binding domain, and, with the other, replacing the C-termmal domain of the polymerase u subunit (aCTDJ. The modified polymerase can be activated by the makeshift "activator" as long as the appropriate DNA-binding site is introduced near the promoter, (n another experiment, the otCTD of polymerase is replaced with a DNA-binding domain (for example, that of CAP). Tins modified polymerase efficiently initiates transcription from the lac promoter in the absence of any activator, as long as the appropriate DNA-binding site is placed nearby. A third experiment is even simpler; polymerase can transcribe the lac genes at high levels in vitro in the absence of any activator if the enzyme is present at high concentration. So we see that either recruiting polymerase artificially or supplying it at a high concentration is sufficient to produce activated levels of expression of the lac genes. These experiments are consistent with the activator having only to help polymerase bind to the promoter. For an explanation of why simply increasing the concentration of a protein (for example, RNA polymerase) helps it bind to a site on DNA (in this case the promoter), see Box 16-5. The results discussed in the box would not be expected if the activator had to induce a specific allostenc change in polymerase to activate transcript/on.

494 Cene Retiulûtiou in Prokaryotas Box 16-2 (Continued)

ONA-binding site

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  • sinit
    Where does the activating region of CAP touch RNA Pol when activating the lac genes?
    4 years ago

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