Harpers

380 / CHAPTER 39AGene for repressor (cl)Gene for CroO RRepressor RNAO R 3 O R 2 O R 1BRepressor promotercro Promotercro RNACT A C C T C T G G C G G T G A TA T G G A G A C C G C C A C T AFigure 39–5. Right operator (O R ) is shown in increasing detail in this series of drawings.The operator is a region of the viral DNA some 80 base pairs long (A). To its left lies thegene encoding lambda repressor (cI), to its right the gene (cro) encoding the regulator proteinCro. When the operator region is enlarged (B), it is seen to include three subregions,O R 1, O R 2, and O R 3, each 17 base pairs long. They are recognition sites to which both repressorand Cro can bind. The recognition sites overlap two promoters—sequences of bases towhich RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines),that are translated into protein. Site O R 1 is enlarged (C) to show its base sequence. Notethat in this region of the λ chromosome, both strands of DNA act as a template for transcription(Chapter 39). (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO:A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)ATtantly, the nucleotide sequences of these three tandemlyarranged sites are similar but not identical (Figure39–5B). The three related cis elements, termed operatorsO R 1, O R 2, and O R 3, can be bound by either cI orCro proteins. However, the relative affinities of cI andCro for each of the sites varies, and this differentialbinding affinity is central to the appropriate operationof the λ phage lytic or lysogenic “molecular switch.”The DNA region between the cro and repressor genesalso contains two promoter sequences that direct thebinding of RNA polymerase in a specified orientation,where it commences transcribing adjacent genes. Onepromoter directs RNA polymerase to transcribe in therightward direction and, thus, to transcribe cro andother distal genes, while the other promoter directs thetranscription of the repressor gene in the leftward direction(Figure 39–5B).The product of the repressor gene, the 236-aminoacid,27 kDa repressor protein, exists as a twodomainmolecule in which the amino terminal domainbinds to operator DNA and the carboxyl terminaldomain promotes the association of one repressorprotein with another to form a dimer. A dimer of repressormolecules binds to operator DNA much moretightly than does the monomeric form (Figure 39–6Ato 39–6C).The product of the cro gene, the 66-amino-acid,9kDa Cro protein, has a single domain but also bindsthe operator DNA more tightly as a dimer (Figure39–6D). The Cro protein’s single domain mediatesboth operator binding and dimerization.In a lysogenic bacterium—ie, a bacterium containinga lambda prophage—the lambda repressor dimer bindspreferentially to O R 1 but in so doing, by a cooperativeinteraction, enhances the binding (by a factor of 10) ofanother repressor dimer to O R 2 (Figure 39–7). Theaffinity of repressor for O R 3 is the least of the three operatorsubregions. The binding of repressor to O R 1 has twomajor effects. The occupation of O R 1 by repressorblocks the binding of RNA polymerase to the rightwardpromoter and in that way prevents expression ofcro. Second, as mentioned above, repressor dimer boundto O R 1 enhances the binding of repressor dimer to O R 2.The binding of repressor to O R 2 has the importantadded effect of enhancing the binding of RNA polymeraseto the leftward promoter that overlaps O R 2 andthereby enhances transcription and subsequent expressionof the repressor gene. This enhancement of transcriptionis apparently mediated through direct proteinproteininteractions between promoter-bound RNApolymerase and O R 2-bound repressor. Thus, the lambdarepressor is both a negative regulator, by preventingtranscription of cro, and a positive regulator, by enhancingtranscription of its own gene, the repressor gene.This dual effect of repressor is responsible for the stablestate of the dormant lambda bacteriophage; not onlydoes the repressor prevent expression of the genes necessaryfor lysis, but it also promotes expression of itself to