DNA structural changes

Changes in the primary, secondary and tertiary structure of DNA can

influence transcription of genes (Zubay, 1980). Primary structure

changes include transient single strand cleavage which is known to encourage transcription ^n vitro and mature T5 phage DNA contains five

single stranded breaks (Zubay, 1980). Base modifications are also

primary structure changes and in T4 phage DNA hydroxymethylation of cytosine is an aid to late transcription (Zubay, 1980). Analogues of cytidine, such as 5 aza-cytidine, resistant to methylation induce changes in the phenotype of mouse embryo cells in culture which develop into striated muscle cells and adipocytes several days after exposure

(Jones and Taylor, 1980). In vitro DNA methylation in the 5' region of

the human gamma globin gene introduced into mouse L cells prevents transcription while methylation in the gamma globin structural regions

cytosine to 5 methylcytosine is involved in the control of gene expression and that DNA modification in general may be involved in development.

Secondary structure changes involve the transition from double

stranded to a single stranded structure. In some viruses such as T4

bacteriophage some late genes are only transcribed where DNA is being

replicated and because this involves the transient appearance of single-

stranded DNA it is possible that some promoters are only recognized in

the single stranded form (Zubay, 1980). It appears that the DNA in

prokaryotic chromosomes is coiled in nucleosome like structures,

although iri vivo only part of the DNA is arranged in this way

(Pettijohn, 1982). The DNA in the isolated nucleoid is negatively

supercoiled and the chromosome segregated into distinct domains of supercoiling such that the torsional tensions can be maintained

independently in each domain (Pettijohn, 1982). Regulation at this

level could provide a basis for co-ordinate control of operons in each

domain. It is known that the introduction of negative supercoils into

DNA stimulates the in vitro template activity. Possibly the explanation

for this is that the formation of rapid start complexes partially

unwinds the double helix introducing positive supercoils. If the DNA is

negatively supercoiled the energy for unwinding the double helix can be

supplied by unwinding the supercoils (Zubay, 1980). It is also possible

that RNA polymerase can more easily enter negatively supercoiled DNA.

The degree of supercoiling of DNA can differentially activate promoters and there are sites on the the DNA separate from promoters where

topoisomerases can act to produce this differential gene expression

(Smith, 1981). Topoisomerase I, also known as omega protein, relaxes

negatively supercoiled DNA. Mutations in the topoisomerase I gene (supX) restore expression of promoter mutations in the leu operon of Salmone11a

typhimurium (Smith, 1981). A possible interpretation of this is that some promoters are active only when the DNA is fully negatively

supercoiled by DNA gyrase. In the absence of topoisomerase DNA remains

negatively supercoiled and the mutant leu operon can be expressed. Inhibitors of DNA gyrase such as nalidixic acid inhibit transcription from some promoters but not others while topoisomerase I can also

prevent expression of some promoters by relaxing the DNA. Some

promoters can be turned on either by a specific activator protein or by

supercoiling. For example the lac operon is activated by a CAP-cAMP

complex and is inactivated by DNA gyrase inhibitors but activated by

topoisomerase I mutations (Smith, 1981). It is possible that the CAP-

cAMP complex activates promoters by unwinding DNA locally while supercoiling achieves this over a whole domain, with DNA gyrase and topoisomerase I acting at particular sites to control the expression of

all the genes in a domain. DNA gyrase synthesis may itself be

controlled by the degree of supercoiling in E^_ coli in a homeostatic mechanism with relaxed DNA being a good template for ^n vitro DNA gyrase

synthesis and supercoiled DNA a poor template (Menzel and Gellert, 1983).

DNA supercoiling and its effect on gene expression may extend to

the eukaryotes. The template surface of DNA is buried within the double

helix and must be exposed during transcription by unpairing bases. Just such an unpairing is induced by negatively supercoiling the DNA. As a result, supercoiled duplexes are cleaved by single strand specific

nucleases and bind single strand specific chemicals. If HeLa cells are

lysed in 2M sodium chloride nucleoids are released which contain nuclear DNA packaged within a flexible cage of RNA and protein (Akrigg and Cook,

1980). The DNA behaves as if it were unbroken and supercoiled.

enzyme from rat liver and DNA gyrase from Escherichia c o l i , can be used

to twist and untwist the nucleoid DNA and alter the degree of exposure

of bases. The supertwisting produced by the action of DNA gyrase

dramatically increases transcription by RNA polymerase II from wheat

germ. In vivo eukaryotic DNA is folded around histone cores to form

nucleosomes and the isolated complex contains no free energy that can be

released by the untwisting enzyme (Akrigg and Cook, 1980). It is

thought that this makes it unlikely that there is a eukaryotic

counterpart to the bacterial DNA gyrase. It is however an attractive

hypothesis to suppose that a eukaryotic gyrase acts to expose the template surface, stimulate transcription and so determine the activity

of genes. Some evidence for the existence of a eukaryotic DNA gyrase in

HeLa cells is provided by the observation that the DNA of cells grown in the presence of novobiocin, a specific inhibitor of the bacterial

gyrase, is less negatively supercoiled than if not treated (Akrigg and

Cook, 1980). This form of topological gene control has received support

recently (Lilley, 1983). One important observation is that the

reactivation of stored yeast mating type gene copies on plasmids which are normally silent is accompanied by alteration of the plasmid

topological state.

1.3.4. DNA dependent RNA polymerase modification and replacement

In Escherichia coli only a single species of DNA dependent RNA

polymerase is to be found. The core enzyme consists of three different

subunits, alpha, beta and beta-prime. Another subunit, the sigma

factor, is needed to give full activity on native DNA. The core enzyme

plus the sigma factor, known as the holoenzyme, has the structure

a2&0'a

the holoenzyme is thus about 500,000 and is one of the most complex enzymes in the bacterial cell (Lathe, 1978). However mitochondrial RNA

polymerase is a single polypeptide of Mr 60,000. The complexity of the

bacterial enzyme probably reflects the need for transcriptional regulation and RNA polymerase may be an allosteric enzyme that can

differentiate between different promoters. Some control factors may

operate by converting the enzyme from one form to another. In

eukaryotes there are three structurally distinct enzymes. Polymerase A

is found in the nucleolus and synthesizes rRNA. Polymerase B is found

in the nucleoplasm and synthesizes heteronuclear (hn) RNA while the

third, polymerase C synthesizes 5S and tRNA. The eukaryotic polymerases

are all heteromultimers with similar but more complex structures to the bacterial enzyme. Like the prokaryotic enzyme the eukaryotic

polymerases probably show allosteric specificity changes with each type having a restricted initiation range (Travers, 1976).

The entire replacement of an RNA polymerase is seen in some

bacteriophage infections. When phage T7 infects coli one of the

early genes transcribed by the host polymerase codes for a viral

polymerase which transcribes the remainder of the viral genome while the host polymerase is inhibited by proteins encoded by the early T7 genes

(Zubay, 1980).

Apart from complete replacement the existing structure of the RNA polymerase could be directly modified so that the specificity for

different promoters is changed. The structural changes could be brought

about by subunit replacement or subunit modification. Several lytic

phages, for example T5, SP01, produce proteins ¿n vivo which bind to the host polymerase and alter the transcriptional specificity (Zubay, 1980;

Do i , 1977). During the formation of virions and endospores in Bacillus

factor proteins that modify the promoter recognition specificity of the

RNA polymerase. In B_^ subtilis the core enzyme can interact with at

least six proteins to achieve a number of different promoter recognition

specificities (Doi, 1977). The growth of phage SP01 in subtilis

involves expression in a temporal sequence controlled at the level of

promoter recognition (Doi, 1977). The early genes are expressed

immediately after infection and are recognised by the unaltered RNA

polymerase containing sigma'’’’. After 5 minutes the middle genes are

expressed by the action of an early protein, gene product 28 or

sigmaSP^®, which binds to the core enzyme and displaces sigma^. In a

similar way late gene transcription is activated by gene products 33 and

34 or sigma acting synergistically. In this way the temporal

programme of gene expression can be explained by a cascade of phage

encoded sigma factors. In order to investigate the promoter sequences

recognised by RNA polymerases with different sigma factors, regions of DNA protected from DNAase I nicking by DNA polymerase were investigated

(Doi, 1977). These regions were 60-70 base pairs long and extended from

40-50 bases preceding the start of RNA synthesis to about 20 bases past the start point (i.e. from -40 to -50 to position +20). The binding

sites recognised by coli RNA polymerase containing the sigma factor

showed 2 regions of homology. From position -30 to position -35 called

the -35 region the most common sequence is 5'TTGACA while from position -12 to -7 called the Pribnow box the most common sequence is 5'TATAAT

(Doi, 1977). The sequence of the SP01 early gene promoter recognised by

B. subtilis RNA polymerase differs from this by only one nucleotide in