Gene structure, organisation and expression

In general, transcriptional signals in genes from higher organisms are more complex than those found in bacterial systems. Within fungi, gene organisation shares many common features across a wide num-ber of genera. The three main organisational units in fungal genes can be split up into those signals that (a) control the switching on or off of gene transcription (promoters), (b) control the termination of transcription (terminators) and (c) control the necessary excision of introns from mRNA.

Unlike their bacterial counterparts, fungal promoters can extend a substantial distance (>1 kb) upstream of the transcriptional start point (tsp). Early cloning experiments in S. cerevisiae suggested that native S. cerevisiae promoters were required for the expression of for-eign genes although subsequent experiments have shown that some promoters from other yeasts, e.g. K. lactis, can also function in S. cere-visiae. In filamentous fungi, promoters tend to function well within their own genus but are less predictable in more distantly related species.

Promoters can generally be defined as either constitutive, i.e. they are switched on permanently, or inducible, i.e. contain elements that

allow them to be switched on or off in a regulated fashion. In both types of promoter, one or more tsp can exist. Sequences in the pro-moter region define both the tsp and the binding sites for regulatory proteins. TA-rich regions, known as TATA-boxes, are often involved in determining the tsp and also maintaining a basal level of transcrip-tion. An illustration of their function is provided by the S. cerevisiae HIS4 promoter where transcription can occur either with or without a TATA-box (the HIS4 gene encodes histidinol dehydrogenase involved in histidine biosynthesis); high transcription levels are only observed from the HIS4 promoter containing a TATA-box. However, there are also strong yeast promoters that lack a TATA-box. Since many yeast and filamentous fungal promoters do not contain this sequence, other motifs, such as the pyrimidine-rich tracts (CT-boxes) found in fila-mentous fungal promoters, can perform the same function. These sequences can be used to describe what is essentially a ‘core’ pro-moter. A third sequence, CCAAT, is also often associated with core promoter function. However, most (∼95%) S. cerevisiae genes appear not to require CCAAT-boxes for function although this motif is known to function in A. nidulans. It should be stressed that in the majority of fungal promoters that have been isolated, the functional signifi-cance of sequences identified in them has not been determined and therefore remains unclear.

With a constitutive promoter, the basal level of transcription is determined by the binding to the core promoter of a protein com-plex which contains RNA polymerase and the so-called general or upstream factors. In contrast, the expression of an inducible gene can change by orders of magnitude. The regulatory elements responsible for mediating this switch are usually found upstream of the core promoter sequences and are termed upstream activation or upstream repression sequences (UAS or URS). These UAS/URS sequences bind regu-latory proteins that are thought to stabilise (or destabilise), either directly or indirectly, the transcriptional complex bound to the core promoter thus elevating/decreasing the rate of transcriptional initiation.

Regulatory sequences which control expression of an inducible promoter may often be found in promoters of several genes that encode proteins of unlinked function, all of which are controlled by a single regulatory protein. Such a network of co-regulated genes may respond to physiological parameters affecting the cell such as pH, carbon or nitrogen source. For example, in the filamentous fungus A. nidulans, a protein (termed PacC) that binds to a specific sequence in the promoters of genes encoding pH-responsive proteins has been identified(Fig. 5.14). When the ambient pH is alkaline, PacC is acti-vated by a protease to a form that permits expression of a wide range of alkaline-expressed genes (e.g. isopenicillin N synthase) and represses many genes normally expressed under acid conditions (e.g.

acid phosphatases). Similarly, in S. cerevisiae and some Kluyveromyces spp., the DNA-binding protein, Mig1p, binds in a glucose-dependent fashion to GC-rich regions (GC-boxes) of many promoters of genes

Alkaline pH

Plasma membrane Signal transduction pathway

PacC

inactive Active

Activation of ‘alkaline’ genes repression of ‘acid’ genes

Figure 5.14 Regulation of transcription by pH in filamentous fungi. The external pH is sensed by cells and leads to changes in gene expression. In fungi, the transcription of genes which encode proteins that are necessary for survival at alkaline pH is mediated by a transcription factor (PacC). Alkaline pH is sensed and leads, through a signal

transduction pathway, to the cleavage of an inactive form of PacC to produce the active form. The activated PacC stimulates the transcription of genes leading to enzymes such as alkaline phosphatase that are active at alkaline pH (‘alkaline’ genes) and represses the transcription of ‘acid’ genes. The truncated form of PacC activates genes by binding to its target promoters at the sequence 5^-GCCARG-3^(where R= G or A).

involved in carbon source utilisation. Mig1p forms a complex with other proteins that represses transcription of genes required for the catabolism of carbon sources which are less efficient in producing energy than glucose and related sugars, a process known as carbon catabolite repression. In filamentous fungi, such as A. nidulans and Trichoderma reesei, a similar function is performed by the CreA and Cre-1 proteins, respectively, which bind to GC-boxes in the promoters of genes involved in carbon catabolism. Another network of genes, whose control is mediated by the regulatory protein AreA in A. nidu-lans (Nit2 in N. crassa), is that involved in the synthesis of enzymes for nitrogen catabolism. In the presence of the preferred simple nitrogen sources, ammonium and glutamine, genes responsible for breaking down complex nitrogen sources are switched off.

In addition to these regulatory systems of broad specificity cov-ering networks of genes, there are also control mechanisms specific to a particular metabolic pathway. Thus, the positive regulatory pro-tein, AflR, co-ordinates the expression of at least 25 different genes responsible for the synthesis of the fungal toxin, aflatoxin. All those genes examined so far contain a specific sequence in their promoters to which the AflR protein binds and activates transcription. Although in this instance the genes are clustered on a small region of one chromosome, there is no known requirement for genes which are co-ordinately regulated (either in a global or pathway-specific context) to be physically contiguous or even be on the same chromosome.

What knowledge there is regarding termination of transcription in fungi has derived mainly from studies with S. cerevisiae where it is

tightly coupled to a process called polyadenylation, that is the addition of long stretches of adenine nucleotides to form polyA mRNA tails.

A major function of polyadenylation is to increase the stability of mRNA; in mutants of S. cerevisiae where the rate of polyA removal is slowed down, mRNA stability is increased. Most yeast genes lack the sequence AATAAA associated with polyadenylation in higher eukary-otes although two other motifs have been associated with termina-tor function: a TTTTTAT motif, which functions only in one orienta-tion, and a tripartite signal based on a TAG . . (T-rich) . . TA(T)GT . . (AT-rich) . . TTT sequence, which can function in either orientation.

It seems likely that a number of signals are used to terminate tran-scription in S. cerevisiae.

The genes of higher organisms also differ from their bacterial counterparts through the presence of introns, which must be excised from the mRNA before it is translated into protein; a process known as splicing. S. cerevisiae and filamentous fungal genes show consid-erable differences with regard to the presence and nature of their introns. The majority of S. cerevisiae genes lack introns altogether and those that do contain introns often contain only one. Genes from filamentous fungi, and those of S. pombe, often have several introns, usually between 50 and 100 bp in size. The fact that the introduc-tion of filamentous fungal genes into S. cerevisiae results in incorrect splicing suggests that the splicing mechanisms differ significantly between some fungal species, although the amdS gene from A. nidu-lans, which has three introns, is spliced correctly in several filamen-tous fungal species. Intron position, though not sequence, is often conserved across filamentous species and introns need not necessarily be within the coding region but can be found in the region between the tsp and the point at which translation of the mRNA starts.