Eukaryotic Transcriptional Activator Essay Example for Free

Eukaryotic Transcriptional Activator EssayIntroduction Much of what was initially known about placement came from pioneering prokaryotic system studies that followed the1959 discovery of RNA polymerase. During those earlier times, it was presumed that gene structure and organisation in bacterium were practically the same for eukaryotes. This later turned out to be incorrect since eucaryotic DNA assumes high-order geomorphologic forms and recordingal and regulative processes in eukaryotes are much more complex. Thus, studies on eukaryotic written text have hold up invaluable in further understanding this vital process that regulates gene expression in higher organisms (The Royal Swedish Academy of Sciences 2). One such pack was done by brent and Ptashne, wherein they investigated which of twain proposed mechanisms does GAL4 part transcription (729). GAL4 is a protein that initiates the transcription of the GAL1 gene in S. cerevisiae, given that a region called UASG or a certain 17-bp sequence (termed 17-mer) is present anywhere from 40 to 600 nucleotides upstream of the genes transcription start site. The two regions bind GAL4 to prompt transcription similarly when inserted upstream in another gene, CYC1 commonly regulated by the two UASs (upstream activation sites) UASC1 and UASC2, which bind certain cellular proteins (in Brent and Ptashne 729). GAL4 is thought to activate transcription either by a) binding to DNA and stabilizing unusual DNA structure so that protein binding near the transcription site is promoted or b) binding to DNA without disturbing its structure and pioneer transcription by getting in contact with other proteins. Based on earlier lambda essays that touch on mutant repressors which, operating via mechanism b above, quarter bind DNA except are in military unitual to activate transcription beca hire the amino acids in the region thought to contract RNA polymerase were altered, Brent and Ptashne essay to determine t he field of operations responsible for GAL4s DNA-binding and activator obligations.For this purpose they used LexA-GAL4, a new protein construct having the DNA-binding specificity of LexA, an E. coli repressor protein whose amino-terminal domain binds to instrument regions to repress gene expression (729). It was found that LexA-GAL4 functions in the same manner in E. coli, but activates transcription in barm if and only if, a lexA operator is also present near the transcription start site (730).Data Analysis The synthesis of LexA-GAL4 in bacteria and yeast was facilitated through the use of plasmids. The gene for LexA-GAL4 is the combination of the E. coli DNA fragment that codes for the 87-residue amino-terminal of LexA, and the S. cerevisiae fragment coding for the 807-residue carboxy-terminal of GAL4. Figure 1a (see gameboards and Figures) shows the DNA sequence and agree amino acids coded in the LexA-GAL4 fusion junction while b and c respectively show plasmid 1109, whos e LexA-GAL4 synthesis is regulated by the tac promoter, and 1027, regulated by the ADH1 promoter (Brent and Ptashne 730). LexA-GAL4s repressor activity in E. coli was demonstrated by two experiments. Table 1 summarizes the results of the first experiment on a bacterial agate line wherein a lacZ gene was adjoined to the lexA promoter. LexA autorepresses its own transcription so the strain used carried a mutant, nonfunctional lexA gene. Plasmids were then used to synthesize different regulatory proteins after which repressor activity was measured by the amount of b-galactosidase produced by lacZ. The results show that LexA-GAL4 transcription repression from the lexA promoter was comparable to that of LexA.Meanwhile, Figure 2 shows the results of the second experiment which made use of the fact that certain LexA-repressed genes need to be expressed for cells to recover from DNA damage. That is wherefore, cells with a mutant LexA that is able to bind to the operator but cant be deact ivated through proteolysis present UV sensitivity. Figure 2 shows the survival rate of E. coli cells depending on the regulatory proteins synthesized by corresponding plasmids. As with the first experiment, LexA-GAL4 showed a similar repressor action as with LexA so that E. coli cells that had them were markedly UV-sensitive compared to cells that had no regulatory protein or had the l repressor which does not recognize the lexA operator and hence has no regulatory effect on transcription (730-731). In contrast to its action in E. coli, LexA-GAL4 acts as a transcriptional activator in yeast when a lexA operator is present. Plasmids were used to transform one group of GAL4+ cells into producing LexA-GAL4 and another to produce native LexA. twain groups were then further modified to carry a gene made from the fusion of either GAL1 or CYC1 and lacZ, and either UASG, the 17-mer, UASC1 and UASC2, a lexA operator, or none of these upstream of the gene (see Figure 3).From the CYC1-lacZ g ene results in Table 2, it can be seen that whereas LexA repressed b-galactosidase production, LexA-GAL4 activated transcription but only when there is a lexA operator upstream. Transcription appeared to be stimulated more when the operator is nearer the transcription start site. Conversely, transcription was markedly hindered in the glucose medium (731-732) which is consistent with previous observations that GAL4 is only active when cells are grown on a galactose medium but is inhibited in the presence of glucose (729). Table 3 shows the same path in LexA-GAL4 activity with the GAL1-lacZ gene.In fact, LexA-GAL4s dependency on the presence of a lexA operator to activate transcription was also emphasized in similar experiments using strains having either a gal4 gene transmit mutation or a gal4 deletion, wherein LexA-GAL4 activated CYC1-lacZ and GAL1-lacZ transcription only when an operator was present and likewise, was dependent on operator proximity to the transcription start site . In these experiments, LexA-GAL4 failed to stimulate b-galactosidase production even in plasmids boot UASG or the GAL1-lacZ gene, nor was it able to compensate for the absence of wild-type GAL4 when no operator was present (731-732).Comparison of LexA-GAL4-stimulated GAL1-lacZ transcription with that in a plasmid bearing wild-type UASG showed that the 5 ends of the RNAs made were the same (Figure 4). However, it is not yet clear why the amount of transcripts produced was only 5% of that which was expected based on b-galactosidase measurements (731).The reduced activity of GAL4 on glucose media is attributed to the acquaintance of the GAL4 C-terminus with the inhibitory protein GAL80, thus hindering efficient binding with UASG (729). The results in Table 4 indicate that the LexA-GAL4 C-terminus likewise associates with GAL80. A glucose medium was used to grow GAL4-producing cells that had UASG but no lexA operator upstream of a GAL1-lacZ gene. Results stir that LexA-GAL4 proteins , in the absence of an operator to bind to, are free to interact with GAL80 and consequently facilitate transcription by leaving wild-type GAL4 to bind to UASG (732-733). Figure 5 shows a spliced yeast gene and a derivative wherein a lexA operator was inserted into the genes intron. This was done to test whether LexA-GAL4 can also activate transcription if the operator is downstream of the normal transcription start site. UASG was present upstream but a gal4 strain was used so no GAL4-stimulated transcription would occur and b-galactosidase production would be purely dependent on LexA-GAL4. From the results in Table 5, it may be seen that LexA-GAL4 was able to stimulate transcription only when theres an operator in the intron, though b-galactosidase production was only 4% as much of that resulting from transcription from UASG in a GAL4+ strain (733). The essence of this studys findings is depicted in Figure 6, which shows that the hybrid protein LexA-GAL4 can successfully stimulate transcription in yeast but only in the presence of a lexA promoter upstream (733). Tables 2 3 and more importantly, the parallel experiments with the GAL4-expression impaired strains (731-732), best illustrate LexA-GAL4s strict requirement for the presence of an operator in order to activate transcription.Conclusions Through the series of experiments done, Brent and Ptashne were able to gather data attributing activator function to GAL4s C-terminus, consequently suggesting that activation by GAL4 is more credibly achieved by its interaction with other proteins rather than by binding to UASG and then perturbing DNA structure. Since LexA-GAL4 successfully activated transcription without binding to UASG, a channelize in structure doesnt appear to be crucial for transcription to occur (733).Though the results of the experiments were per se quite conclusive, they are rather indirect consequence for the GAL4 mechanism being put forward. A probably more direct proof is offered by the K eegan, gill and Ptashne study mentioned which claims that another hybrid protein having the amino terminal of GAL4 binds UASG but fails to activate transcription, likely because the C-terminus is that of b-galactosidase which functions differently (733).This study has successfully illustrated the synthesis of hybrid proteins that can be used for exploring further not just the activator function of other eukaryotic regulatory proteins (734), but on the whole, transcriptional and regulatory processes in various other eukaryotic organisms.Good follow-up studies would therefore be a structural study to determine whether no change in DNA structure is indeed involved in GAL4 activity and more generally, the application of the methods and concepts erudite here to other eukaryotic genes and their known regulators so as to perhaps be able to point whether a mechanism similar to that proposed for GAL4 is also in play. Both ultimately can help to pass water a general but detailed picture t hat will allow for a deeper understanding of eukaryotic transcription and regulation of gene expression.