replication-based recombination so have to rely on processes of direct genomic change to enable diversity in their populations. Microorganisms face many environmental fac- tors that could possibly cause mutation in their genomic DNA. Permanent changes in the DNA caused by external factors are known as induced mutagenesis. Those changes caused by errors in the DNA replication machinery, or other mistakes made by the cel- lular DNA metabolizing enzymes are known as spontaneous mutagenesis. The rate of error in DNA replication is between 10-7 and 10-11 per base pair per round of replication,
equivalent to around 10-4 to 10-8 errors per gene, per generation in a bacterium the size
of E. coli. Spontaneous change can also include those genomic rearrangements that an organism may make to its own genome, as exhibited by Streptomyces. These mutation
hot spots on the genome are normally associated with an abundance of short inverted
repeats. It is thought that the repetitive nature of the sequence causes polymerase to stut- ter and make more errors at these particular points.
A strain isolated from its environment and held in pure culture in the laboratory is defined as the wild type of a particular strain. Any strain derived from the wild type with any change in its genomic make up compared with this wild type is known as a mutant. This change can be referred to as a change in genotype. If the difference in genotype results in an observable change to the properties of the organism, such as a sudden inability to use a particular carbon source, this is said to be a change in phenotype. Changes in depen- dence on medium components are frequently used in the elucidation of biochemical pathways or in the deduction of regulatory systems, so the mutation of the wild type with a nutritional requirement is a common experiment. These strains are known as auxo-
trophs. For example, if E. coli is subjected to a chemical mutagen and as a result of muta-
tion now requires the vitamin B12 to be present in its medium for the strain to grow, the strain is auxotrophic for B12, or can be called a B12 auxotroph.
Key Notes
Transcription The first stage of converting the primary genetic information from the stable DNA code into protein is to transcribe the DNA into messenger RNA (mRNA). The RNA polymerases that carry this out bind to a DNA-coded signal (promoter) upstream of the gene to be transcribed. Genes in Bacteria can be transcribed alone (monocistronically) or with others as part of a polycistronic operon.
Promoters RNA polymerase is composed of four polypeptides (abb¢s). The core polymerase (abb¢) has a high processivity but low DNA affinity. The presence of sigma factor (s) makes the holo-polymerase and confers DNA sequence specificity to the promoter. Once the RNA polymerase has bound, the sigma factor dissociates. The core polymerase then synthesizes RNA complementary to the lower (template) DNA strand. In E. coli, the promoter is made up of two conserved regions, the ‘–10’ or Pribnow box, and ‘–35’. The numbers –10 and –35 refer to the number of bases the sequence is from the base where transcription starts. The –10(TATAAT)/–35(TTGACA) consensus promoter is not the only sequence of promoter in the genome, other sequences allow the binding of alternative sigma factors, which can control specialized groups of genes. These regulons can thus be switched on and off according to external changes in the cell’s environment.
Termination of The signal for termination of transcription is provided by
transcription a structure on the mRNA itself. Termination can be signaled by a stem-loop structure, or by the action of the protein Rho.
Regulation of All genes are regulated in some way at some stage during the
transcription cell cycle. In Bacteria this mainly happens in two ways: by derepression (where a protein bound to a promoter stopping transcription is removed and the gene is switched on) and attenuation (where the presence or absence of a substrate necessary for the function of the gene product governs the transcription of the gene itself). Less common is activation, where the presence of protein is used to switch a gene on.
The lac operon The lac operon is made up of a promoter, an operator (a site on the DNA where regulatory proteins bind) and the genes lacZ, lacY, and lacA. Another promoter controls the transcription of lacI, coding for the regulator (Lac repressor protein). When LacI is bound to the lacZYA operon operator, transcription is blocked and the cell is unable to produce b-galactosidase. Allolactose (a by-product of the action
125
F4 – TRANSCRIPTIONof b-galactosidase on lactose) is the primary inducer of the operon. In this derepressed state, the lac promoter is relatively weak and only achieves full strength if the protein CRP binds as well.
The trp operon The tryptophan (trp) operon is made up of five genes (trpEDCBA), regulated by the binding of the trp repressor complexed to tryptophan. A series of stem-loop structures can form, which regulate transcription of the operon by attenuation.
Related topics (E2) Electron transport, (F5) Messenger RNA and oxidative phosphorylation, translation
and b-oxidation of fatty (F6) Signal transduction and
acids environmental sensing
transcription
The first stage of converting the primary genetic information from the stable DNA code into protein is to transcribe the DNA into messenger RNA (mRNA). This process is car- ried out by RNA polymerase, which has many common features with the DNA polymer- ases. The structure of the RNA polymerase is a little similar to the DNA polymerases in the active site region, and also requires Mg2+ to function and synthesizes nucleic acid in a
5¢ to 3¢ direction. However, the RNA polymerases do not require a primer to initiate syn- thesis. Instead, their signal for the initiation of transcription is a specific sequence on the DNA, known as the promoter region.
A gene’s promoter is said to be ‘upstream’, in that the promoter region is situated to the 5¢ end of the coding region. The promoter region allows the RNA polymerase to bind and begin transcription so that the resulting mRNA contains not only the coding region itself but also all the signals to start and stop the synthesis of the polypeptide. How RNA polymerase works is intrinsic to the concept that one gene makes one polypeptide. In eukaryotes, genes are arranged so that the promoter region is in such a position that when transcription occurs a single mRNA molecule is produced that can be used to code for a single polypeptide (monocistronic). Genes that code for polypeptides that have a common purpose (such as the manufacture of a multi-polypeptide protein) are placed in many different parts of the genome, frequently on different chromosomes. In prokary- otes (both the Bacteria and the Archaea) genes are more likely to be arranged so that the coding regions for enzymes involved in a single pathway are clustered together. Further- more, several genes may be arranged so close to one another that they are transcribed from a single promoter (Figure 1). This polycistronic arrangement is called an operon (see the lac operon below).
Promoters
RNA polymerase is composed of four different polypeptides, called beta, beta prime, alpha, and the sigma factor (abb¢s). The core polymerase (abb¢) has a high processiv- ity (RNA polymerizing activity) but low DNA affinity. The addition of the sigma factor to make holo-polymerase confers DNA sequence specificity, forcing the RNA polymerase to bind at the promoter region. Once the RNA polymerase has bound, the sigma fac- tor dissociates. The core polymerase then synthesizes RNA complementary to the lower
(template) DNA strand. The promoter region tends to have a slightly higher A and T con- tent than the surrounding DNA, and the most abundant form of promoter in Escherichia
coli has two fairly well conserved sequences, TATAAT (the Pribnow box or –10 sequence)
and TTGACA (the –35 sequence). The higher A/T content means that there are fewer hydrogen bonds to hold the double-stranded DNA together and thus the double helix is easier to force apart, a process termed melting. Melting of the promoter region allows access of the RNA polymerase, which specifically targets the –10 and –35 regions by the use of the sigma factor. The numbers –10 and –35 refer to relative position in relation to the number of bases the sequence is from the base where transcription starts (known as the transcription start site, Figure 2).
The –10(TATAAT)/–35(TTGACA) consensus promoter is not the only sequence of pro- moter in the genome. There are many promoters with a sequence very similar to –10/–35 and the greater the difference in the base sequence is to this consensus, the weaker the promoter. A strong promoter (such as that of the lac operon) forms a very tight bond with the sigma factor, and transcription is very likely to be initiated from such a promoter. A weak promoter binds the sigma factor by only a few bases, and is concomitantly less likely to initiate RNA synthesis. Other promoters have completely different sequences that bind alternative sigma factors. A good example of this is the alternative sigma fac- tor produced in response to low oxygen in E. coli and some other bacteria. The sigma factor still allows RNA polymerase to recognize a –10 site, but instead will only allow the holo-polymerase to bind where there is an additional specific sequence (FNR site) at around 42 bases upstream of the transcription start site. The use of alternative sigma fac- tors allows a whole group of genes and operons, known as a regulon, to be switched on and off according to external changes in the cell’s environment (Section F6). Induction of the FNR regulon allows the cell to induce all the genes that are useful to cope with low oxygen, principally alternative electron acceptors (Section E2).