4.1. ANALISIS Y CARACTERIZACION DEL LUGAR DE ESTUDIO
4.1.14. ATRACTIVOS TURISTICOS
The study of how the heat shock response is regulated has provided a great deal of information about the molecular mechanisms involved in mounting a transient global response to an environmental stimulus. It is apparent that the response is orchestrated in such a way as to suit the specific requirements o f different organisms. In Drosophila,
regulation is exerted on both transcription and translation (Lindquist, 1981). This is not surprising given that mRNAs in Drosophila have half-lives of between 6-9 hours. To effect rapid changes in protein synthesis the cells must therefore block translation of pre-existing mRNAs. E. coli does not have this problem, consequently the response is controlled
Figure 1.4 M odel for autoreguiation o f the heat shock response in E . c o l i . 3 0 C H o u s e k e e p i n g g e n e s A D P + P I A T P 5 - 6 0 m i n u t e s a f t e r h e a t s h o c k 0 - 5 I m i n u t e s . a f t e r A h e a t s h o c k A T P
□
A D P + P i L e v e l s o f h s p s i n c r e a s e 4 2 V H e a t s h o c k g e n e s K E Y : h s p s c e l l u l a r p r o t e i nm
R N A p o l y m e r a s e m i n u s I a f a c t o r 1 D n a K p r o p e r l y f o l d e d0
D n a J U V W a b e r r a n t □ ^ G r p EFree I)naK binds 0 ^ 2 sequestering it from R NA polym erase and/or presenting it to proteases. The 1 1 /2 o f o32 in creases on heat shock due to a com bin ation o f factors, including the association o f DnaK with aberrant protein. T h is allo w s the transcription factor to bind to R N A polym erase w hich lead s to transcription from heat sh ock promoters. I'he ensuing increase o f free DnaK levels lead to the recapture o f thereby re-establishing low levels o f it, thereby dam pening the heat shock response. This figure is com piled from data found in the references eited in section 1.3.1. R ecent experim ents have show n that purified DnaK can bind to the punfied o factor (Libcrek et al., cited in Hightower, 1991) hence the direct interaction between the tw o proteins show n above.
almost entirely at the level of gene transcription (Yamamori and Yura, 1980). In addition^ there are examples of regulation arranged in such a way as to satisfy requirements of different cell types within the same organism. Regulation is primarily transcriptional in the somatic cells of Xenopus but translational in oocytes (Bienz and Gurdon, 1982). Due to their enormous size oocytes would require 10-100 days to synthesize an effective amount of heat shock mRNA. The hspTO gene is therefore constitutively transcribed but translationally repressed. On heat shock the tranalational repression is lifted.
1.3.1 Regulation of transcription in prokarvotes.
In E. coli the heat shock response is transcriptionally regulated by the cellular concentration of o32 , a sigma factor that binds to core RNA polymerase and redirects it to heat shock promoters (Grossman et al., 1984). The fact that the promoters for heat shock genes are recognised by this complex, rather than by the predom inant polym erase (containing a^o), allows their regulation to be distinct from the vast m ajority of E. coli
genes. Ordinarily, "housekeeping" genes are transcribed only because levels of o32 (encoded by rpoH) are negatively regulated. Even though rpoH mRNA is relatively abundant, o32 is synthesised at a very low rate (Gross et al, 1990). Also, this protein has a very short half life (ti/2), ca. 45 seconds (Straus et al, 1987).
The intracellular concentration of a32 increases 15 to 20 fold five minutes after a 300C to 42 OC upshift; levels declining thereafter. This is achieved by increased transcription of rpoH, and transient stabilisation of both rpoH mRNA and the product o32 (Gross et al, 1990). This results in preferential transcription of hsp genes. It was anticipated that the transient nature of the response could be coupled to the function of hsps in proteolysis or the control of protein structure. Not surprisingly mutants in dnaK, dnaJ
and grpE fail to switch off the heat shock response (Tilly et al, 1983; Gross et al, 1990). This was due to a defect in degradation of o32. in agreement with this, overproduction of DnaK leads to a dampening of the heat shock response (Tilly et al, 1983). These hsps therefore regulate their own synthesis, and the synthesis of other hsps, by increasing o32 degradation thereby re-establishing the low levels of a32 in the cell (see Figure 1.4).
Though some of the players in this negative regulatory loop are known, the proteins that degrade a32, the mechanism by which t i/2 o f c^2 is increased and the putative translational repressor o f rpoH m RNA, remain unidentified. In addition to this, the discovery of another sigma factor (o24or aE) means that the regulation o f the prokaryotic heat shock response may be more complex than previously thought. The heat shock gene
htrA whose product is essential for E. coli viability at high temperatures (Strauch et a l.,
1989) is under the exclusive control of o24 (Erickson and Gross, 1989) Also, one of the four promoters of rpoH itself is recognised by a24, the induction it elicits being particularly
strong when cells are shifted to 50^X1! (Erickson and Gross, 1989).
1.3.2 Regulation of transcription in eukaryotes.
The induction of eukaryotic heat-shock genes in response to temperature upshift is mediated by the binding of a transcriptional activator, heat shock factor (HSF), to a short highly-conserved DNA sequence known as the heat shock elem ent (HSE). HSEs are defined as an array of a variable number of the five bp sequence nOAAn arranged in alternating orientations upstream of the TATA box (for review see Sorger, 1991) . A t least two nOAAn units are needed for high affinity binding of HSF; these may be arranged head to head (nOAAnnTTCn) or tail to tail (nTTCnnOAAn) (Perisic et al., 1989).
Upon heat shock a pre-existing pool of inactivated HSF is converted to a form capable of stimulating transcription. In S. cerevisiae and K. lactis (budding yeasts) HSF is bound to DNA both before and during heat shock (Jacobsen and Pelham, 1991). HSF becomes highly phosphorylated on heat shock, the transcriptional activity of HSF closely following the extent o f its phosphorylation over a range o f temperatures (Sorger and Pelham, 1988). In contrast, the HSF of &^pombe (fission yeast). Drosophila, and humans binds to DNA only after heat shock. As in S. cerevisiae, this is accom panied by phosphorylation of the factor (Gallo et al., 1991). In higher eukaryotes binding of HSF to the HSF per se appears to be insufficient for transcriptional activation. In murine erythroid - leukemia cells heat shock does not induce hspVO transcription. Though HSF binds to DNA after the temperature upshift, it does not become phosphorylated in these cells (Hensold et a l , 1990).
The kinase responsible for HSF phosphorylation rem ains u nidentified. Alternatively, elevated temperatures may stimulate HSF autophosphorylation. Even though the factor has been purified to homogeneity from several organisms, there have been no reports in the literature ascribing such an activity to it.
In S. cerevisiae the HSF gene is essential for viability (Sorger and Pelham, 1988).
This is thought to be due to the requirement of basal levels of hsps during normal temperatures. There are several lines of evidence for this HSF binding sites in the promoter of one of hsp70 genes mediates 80% of non heat shock (basal) activity (Park and Craig, 1989). Also, overexpressing HSF in the absence of heat shock results in a four fold increase of the level of a major species of hsp70 (Sorger and Pelham, 1988). The sustained basal and transient heat-shock inducible-activities of HSF appear to be m ediated by physically separatable regions of the polypeptide (Sorger, 1990).