Dale_Molecular Genetics of Bacteria 4th ed

Enzyme

Inhibition/activation

activity

Figure 3.1 Information flow and regulatory factors

(1)The number of copies of the gene. In general, if there are several copies of a gene the level of product is likely to be higher (although the relationship is not necessarily linear).

(2)The efficiency with which the gene is transcribed, which is mainly determined by the level of initiation of transcription by RNA polymerase (promoter activity). In bacteria this is the major factor influencing the expression of individual genes, whether we are considering fixed or variable controls.

(3)The stability of the mRNA. It is important to recognize that the amount of specific mRNA will be determined by the combined effect of the rate at which it is

produced and the length of time each molecule persists in an active state in the cell. Most bacterial mRNA is very short-lived, typically being degraded with a half-life of about 2 min. The instability of bacterial mRNA is a key feature in the rapidity with which bacteria can respond to changes in their environment. However, some bacterial mRNA species are more stable than others, in some cases with a half-life as long as 25 min. Other forms of RNA (rRNA, tRNA) are also considerably more stable, which can be ascribed to the high degree of secondary structure possessed by these molecules.

(4)The efficiency with which the mRNA is translated into protein. This will be influenced by the efficiency of initiation (ribosome binding) and also by factors that affect the rate of translation (mainly codon usage).

(5)The stability of the protein product. As with the mRNA, the amount of protein reflects both its rate of production and its stability. Different proteins vary in their stability to a very marked degree, as might be expected from their different functions: a protein that forms part of a cellular structure is likely to be more stable than one that transmits a signal for switching on a transient cellular event.

(6)Post-translational effects. This includes a wide variety of events such as protein folding which is necessary for conversion of polypeptide chains into

a biologically active conformation, as well as covalent modifications that can influence the activity of the protein. Phosphorylation is especially important as a mechanism for regulating the function of specific proteins. In addition, to complete the picture, control of the metabolism and physiology of the cell is influenced by temporary effects such as inhibition and activation of individual enzymes.

We can now consider some of these factors in more detail.

3.1 Gene copy number

Most genes on the bacterial chromosome are present as single copies (with a few notable exceptions such as the genes for ribosomal RNA); gene copy number is not therefore an important method of control for most of the normal metabolic activities of a bacterial cell. It does become important however when we consider plasmid-mediated characteristics, particularly with reference to the cloning and expression of heterologous DNA. Some plasmids are present within the cell in very high numbers, running into thousands of copies (see Chapter 5) and this is reflected in the enhanced level of expression of the genes they carry.

Not all coordinately controlled genes are arranged in operons. In some cases, groups of genes at different sites on the chromosome are regulated in a concerted fashion. Such a set of genes or operons, expressed from separate promoter sites but controlled by the same regulatory molecule is called a regulon (Figure 3.5). For example, arginine biosynthesis requires eight genes (argA-H), but (in E. coli) only three of these (argC, argB, argH) form an operon with a single promoter. A fourth gene (argE) is divergently transcribed from an adjacent promoter (thus providing another example of a divergent operon as described above) while the remaining three genes (argA, argF, and argG) are found at different sites on the chromosome, each with its own promoter. Despite this scattering of the arginine biosynthesis genes, they are coordinately controlled.

Alternative promoters and s-factors

We saw in Chapter 1 that RNA polymerase consists of a core protein, composed of four subunits (a2bb0) with a fifth dissociable subunit called a s-factor (sigmafactor). It is the s-factor that allows the polymerase to recognize specifically the two conserved nucleotide motifs in the promoter region. Thus the sigma factor determines the specificity of the enzyme.

The promoter consensus described above is recognized by the primary s-factor, commonly referred to as s70 (because it is about 70 kDa in size in E. coli). This subunit is responsible for recognition of the promoters used for transcription of most of the genes required in exponentially growing cells. These are sometimes called ‘housekeeping’ genes since they encode essential functions needed for the cell cycle and for normal metabolism such as glycolysis, the TCA cycle and DNA replication. Most bacterial species, however, have several different s-factors. Replacement of the primary s-factor with a different subunit radically changes the recognition of promoter sequences by the RNA polymerase (Figure 3.6). These alternative s-factors allow the bacterium to bring about global changes in gene expression in response to particular environmental stresses. For example, in many bacteria, an abrupt temperature increase triggers the expression of heatshock proteins which can counter the detrimental effects of high temperature. The promoters of about 30 heat shock genes are only recognized by RNA polymerase containing s32 (also called the heat shock s-factor). This normally has a short half-life but following exposure to high temperatures, it is not degraded and thus stimulates transcription of target genes. Consequently, this s-factor allows the bacterium to express a subset of genes whose products are required to counter heat stress. Because many of these genes are scattered around the genome, this system constitutes a heat shock regulon. Other examples of s-factors, their regulatory activity and recognition sequences are given in Figure 3.6. We will encounter further examples in Chapter 4.

Figure 3.6 Alternative sigma factors and promoter recognition sequences. *In B. subtilis. All others are in E. coli

When the Gram-positive bacterium B. subtilis encounters stress, one of the survival mechanisms it can evoke is sporulation, resulting in highly resistant endospores. This is governed by a complex transcriptional regulatory programme that controls the expression of more than 100 genes and involves the sequential activation of six different s-factors. A simplified view is shown in Figure 3.7. At the onset of starvation, the primary s-factor (sA) and a low abundance factor called sH direct the transcription of a set of genes whose products cause an asymmetric invagination of the membrane, thus separating the forespore from the mother cell. During this process a copy of the chromosome is partitioned into each of the two compartments. Another s-factor, sF, is present before the septum forms, but is inactive (due to the presence of an anti-s-factor, see below). When the septum forms, sF becomes active but only in the forespore, where it switches on a new set of genes. Following this, a third sporulation specific s-factor, sE, also becomes active, but only in the mother cell. A different set of genes is thus activated in the mother cell. The forespore then becomes surrounded by a second membrane (engulfment). The subsequent condensation of the spore chromosome is dependent on gene expression mediated by yet another sigma factor, sG, which becomes active in the forespore following engulfment. The changes in the forespore that are brought about by the genes expressed using sG lead to the activation of a further sigma factor, sK, in the mother cell. The products of the

Wall formation; σG spore DNA

condenses

Release of mature spore

Figure 3.7 Sporulation in Bacillus. The diagram shows the stages at which each sigma factor first becomes active

genes expressed at this stage are involved in the final stages of sporulation including the production of the cortex and coat layers that encase the spore, leading to a structure that is highly resistant to desiccation and also to chemical agents such as disinfectants.