Dale_Molecular Genetics of Bacteria 4th ed

An important and intriguing part of this process is that the two compartments of the cell have to develop in parallel. Each stage in the mother cell has to be matched by corresponding stages in the forespore and vice versa. Without this co-operation, the proper development of the spore would not occur. As each stage develops, there is a form of signalling between the two compartments, known as cross-talking, that stimulates the development of the next stage. This provides one of the best understood examples of developmental control in bacterial systems.

Anti-s-factors

The ability of s-factors to activate sets of genes in response to various stimuli has to be controlled so that they are inactive in the absence of that stimulus. One way of doing this (as in the example of sF above) involves anti-s-factors – proteins that bind to a specific s-factor and prevent it interacting with the core RNA polymerase. Anti-s-factors also provide an additional layer of transcriptional regulation as demonstrated by the regulation of flagella biosynthesis in Salmonella typhimurium (Figure 3.8). The flagellum is amongst the most complicated structures built by bacterial cells and requires the coordinated expression of more than 52 genes (1.5 per cent of the genome). One of the major regulators is the flagella-specific s-factor s28. Bacterial flagellar assembly occurs sequentially, with the inner substructures being laid down first, followed by the construction of the external structures. Consequently, one of the problems faced by a bacterium is how to sense when the inner substructures have been completed so that it can start production of the external filament which is attached to this. The FlgM anti-s-factor provides a mechanism for doing just this. Before completion of the inner substructures, FlgM binds to s28 to prevent its action and thus prevent filament production. However, once the intracellular portion (hook-basal body) of the flagella is completed, it serves as a hollow channel and export pathway, not only for the filament protein but also for FlgM. Thus, at the same time as the hook-basal body is completed, FlgM is exported out of the cell and in its absence, s28 can activate expression of the filament genes.

3. 2. 2 Terminators, attenuators and anti-terminators

The structure and action of transcriptional terminators, which stop transcription at the end of an operon, was described in Chapter 1. Transcriptional termination can also play a role in adjusting the level of expression of different genes within an operon.

The genes forming an operon are transcribed from a single promoter and are therefore switched on or off simultaneously. However, although the cell requires all these products at the same time, it does not necessarily need all of

(a) Before construction of the hook basal body, FlgM binds to σ28 to prevent it binding to RNA polymerase (RNAP)

RNAP

σ28

FlgM

(b) After construction of the hook basal body FlgM is exported through a pore formed by the basal body. In its absence, σ28 is able to bind to

RNA polymerase and initiates expression of the flagella filament protein FliC

RNAP

σ28

FlgM

Figure 3.8 The regulation of flagella filament production (FliC) by the anti sigmafactor FlgM

them at the same level. Transcriptional termination provides one way of doing this.

In the example shown in Figure 3.9, the products of genes a and b are required at a higher level than those of genes c and d. The presence of a termination site (labelled t1) located between genes b and c will cause the RNA polymerase to pause. In this case, however, termination will only occur in a proportion of transcripts, while in the remainder transcription will be resumed. This will

is the same: binding of the repressor to the operator prevents the RNA polymerase from obtaining access to the promoter or from initiating transcription.

The lac repressor is a multimeric protein, consisting of four identical subunits, showing a secondary structure feature consisting of two a-helices separated by a few amino acids that place the two a-helices at a defined angle to each other. This conformation, known as a helix-turn-helix motif, is characteristic of DNA-binding proteins and enables this part of the protein to fit into the major groove of the DNA and make specific contacts with the operator DNA. The lac operator site exhibits dyad symmetry. In other words, part of the sequence (shown boxed in Figure 3.10) is repeated (imperfectly) in the inverse orientation. The tetrameric structure of the regulatory protein enables each half of it to recognize half of the binding site, thus increasing both specificity and affinity for the operator (Figure 3.11).

The lac repressor protein also has affinity for allolactose (a derivative of lactose). Binding of allolactose to the repressor causes an allosteric change in the protein so that it is no longer able to bind to the operator site. So, in the absence of lactose, the repressor is active, binds to the operator and prevents transcription of the lac operon (Figure 3.12). In the presence of lactose, the repressor is inactivated, the operon is expressed, and the cell produces the enzymes that are necessary for the metabolism of lactose.

The natural situation in E. coli is therefore that lactose fermentation is an inducible characteristic, i.e. it is only expressed in the presence of lactose. In the laboratory, lactose is an inconvenient inducer, since it will start to be metabolized as soon as induction is effective, thus tending to diminish the effect. However, the ability to act as an inducer and the ability to act as a substrate are separate characteristics: the former depends on recognition by the repressor protein and the latter depends on recognition (and breakdown) by b-galactosidase. It is therefore possible to design analogues of lactose that can still act as inducers, since

Dimerization Repressor domain protein

dimer

DNA-binding domain

Dyad symmetry in operator site

Figure 3.11 Binding of a dimeric regulatory protein to an operator site with dyad symmetry

(a) No inducer

Repressor binds to operator.

No transcription

Repressor

(b) Inducer present

Repressor

Permease

Inducer

Modified repressor unable to bind

to operator

Figure 3.12 Regulation of the lac operon

they will bind to the repressor, but cannot be destroyed by the b-galactosidase. One such gratuitous inducer is the synthetic analogue iso-propyl-thiogalactoside (IPTG).

The converse is also true: some compounds are substrates for breakdown by b-galactosidase but are not able to act as inducers since they are not recognized by the repressor. The chromogenic substrate commonly known as X-gal (which gives a blue colour after hydrolysis by b-galactosidase) is an example of a substrate that does not act as an inducer. Therefore in order to obtain blue colonies it is necessary to add an inducing agent such as IPTG to the medium.

Regulatory mutants of the lac operon

Much of the evidence for the above model is derived from studies of regulatory mutants of the lac operon. These fall into several categories.

Operator

CRP binding

site

Promoter

Figure 3.16 Protein binding sites in the regulatory region of the lac operon

CRP

CRP binding site

Figure 3.17 The cAMP–CRP complex causes DNA bending

interacts directly with RNA polymerase to promote binding of the latter enzyme to the promoter (Figure 3.17). In the absence of the cAMP–CRP complex, RNA polymerase binds very weakly to the lac promoter. Despite the term catabolite repression, it should be clear that the role of the CRP is a positive one; it is an activator (when bound to cAMP) not a repressor. Hence, some people prefer to call it catabolite activator protein (CAP).

An additional effect of the binding of the cAMP–CRP complex is that the DNA becomes bent at this point (Figure 3.17). Many regulatory proteins bend DNA which has local affects on the winding of the helix and may make the promoter more accessible to the RNA polymerase.

Many other operons are sensitive to catabolite regulation involving the cAMP– CRP complex, although details such as the position of the binding site vary