Hi, I need help interpreting this experiment.
The locations of the TATA box in two species of yeast, Schisosaccharomyces
pombe and Saccharomyces cerevisiae differ dramatically. The TATA box of S.
pombe is about 30 nucleotides upstream of the start site, similar to the location in most other eukaryotic cells. However, the TATA box of S. cerevisiae is 40 to 120 nucleotides upstream of the start site. To better understand what establishes the transcriptional start site in these two
species, researchers conducted a series of experiments to determine which
components of the transcription apparatus of these two species could be
interchanged. In these experiments, different transcription factors and RNA
polymerases were combined, and the effects of the switch on the level of RNA synthesis and on the start point of transcription were observed. The results from one set of experiments are shown in the table. Components preceded with a “c” are factors from S. cerevisiae. Components preceded with a “p” are factors from S. pombe. The table indicates whether the components were present (+) or absent (-) in the experiment. In the gel, the presence of a band indicates that RNA was produced and the position of the band indicates whether it was the length predicted when transcription begins 30 bp downstream from the TATA box or from 40 to 120 bp downstream from the TATA box. The gel has larger molecules at the top, and shorter molecules toward the bottom. The table aligns with the gel, and indicates what is present in each reaction.
Experiment (gel lane #)
Components 1 2 3 4 5 6 7
cTFIIE + - + + + + -
cTFIIH + cTFIIF + - + + + - +
cTFIIB + cPol II + - - - - - -
pPol II - + + + - + +
pTFIIB - + + - + + +
pTFIIE + pTFIIH + pTFIIF - + - - - - -
Gel electrophoresis bands
30 bp TATTA box. = =
40 to 120 bp =
TATTA box
The question asks about conclusion regarding the components determine the start site for transcription? I am confused on how some of these transcription components or polymerases working and not working in the some trials. I think the 40 to 120 bp segment is smaller thus farther down compared to the 30 bp segment.
Thanks!
From the provided information, the experiment aimed to determine which components of the transcription apparatus in two yeast species, Schizosaccharomyces pombe and Saccharomyces cerevisiae, are responsible for establishing the transcriptional start site. The TATA box, which is a DNA sequence that helps initiate transcription, is located differently in these two species. In S. pombe, the TATA box is located about 30 nucleotides upstream of the start site, which is similar to most other eukaryotic cells. However, in S. cerevisiae, the TATA box is located 40 to 120 nucleotides upstream of the start site.
To investigate the role of different components in determining the start site for transcription, the researchers conducted a series of experiments. They manipulated the presence or absence of various transcription factors and RNA polymerases and observed the effects on the level of RNA synthesis and the start point of transcription. The results of one set of experiments are shown in the table.
In the gel electrophoresis bands, the presence of a band indicates that RNA was produced, and the position of the band indicates whether it was the length predicted when transcription begins 30 base pairs (bp) downstream from the TATA box or 40 to 120 bp downstream from the TATA box. The gel has larger molecules at the top and shorter molecules toward the bottom.
Now, let's analyze the results in the table:
- Experiment 1: cTFIIE is present (+), and the gel shows a band at the expected length of the 30 bp downstream transcription.
- Experiment 2: cTFIIE is absent (-), and there is no band observed, indicating no transcription occurred.
- Experiment 3: cTFIIH and cTFIIF are present, and a band is observed at the expected length of the 30 bp downstream transcription.
- Experiment 4: In this case, cTFIIH and cTFIIF are present (+), but cTFIIB and cPol II are absent (-). No band is observed, indicating that the absence of cTFIIB and cPol II prevents transcription.
- Experiment 5: cTFIIE, cTFIIB, and cPol II are absent (-), but cTFIIH and cTFIIF are present. A band is observed at the expected length of the 30 bp downstream transcription.
- Experiment 6: pPol II is present (+), and a band is observed at the expected length of the 40 to 120 bp downstream transcription.
- Experiment 7: pTFIIB is present (+), but pTFIIE, pTFIIH, and pTFIIF are absent (-). No band is observed, indicating the absence of pTFIIE, pTFIIH, and pTFIIF prevents transcription.
Based on these results, we can draw some conclusions regarding the components' role in determining the start site for transcription:
- cTFIIE is important for transcription initiation at both the 30 and 40 to 120 bp downstream sites.
- cTFIIH and cTFIIF are also critical for transcription initiation at the 30 bp downstream site.
- cTFIIB and cPol II are required for transcription initiation at the 30 bp downstream site.
- pPol II is capable of initiating transcription at the 40 to 120 bp downstream site but not the 30 bp downstream site.
- pTFIIB alone is insufficient for transcription initiation at either site, as observed in experiment 7.
In summary, the presence or absence of specific components determines the start site for transcription. The experiments demonstrated that different combinations of transcription factors and RNA polymerases have different effects on the level of RNA synthesis and the start point of transcription.