A) Which of the transcription components must be present during the preincubation to keep the template active during chromatin assembly? Explain your answer. B) Which of the transcription components form a complex with the template that is stable to chromatin formation and subsequent chromatin purification? Explain your answer. C) Which of the transcription components must be added during the assay in order to produce a transcript? Explain your answer.

Human Anatomy & Physiology (11th Edition)
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Chapter1: The Human Body: An Orientation
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Question 2: You decide to test how the presence or absence of nucleosomes affects transcription using an in
vitro transcription assay in which a DNA template featuring a promoter and single reporter gene is transcribed
with the addition of specific factors in a test tube. You get robust transcription when you combine template
DNA, RNA Polymerase, NTPs, and four general transcription factors - TFIIA, TFIIB, TFIID, and TFIIE.
However, if you assemble the template onto purified nucleosomes and purify the chromatin template, then you
don't get transcription even when all of the transcription components have been added.
You decide to try a slightly different experiment where you first preincubate the template with all the
transcription components except NTPs, then assemble the template onto nucleosomes and purify the chromatin
template. Now when you add the transcription components and NTPs, transcription proceeds just as well as it
does when using the naked DNA template (compare lanes 1 and 3). You conclude that one or more of the
transcription factors must bind to the template and keep the promoter accessible.
To investigate this phenomena in more detail, you carry out two additional kinds of experiments. In one, you
leave out individual transcription factors during the preincubation (lanes 4 – 8). In the second, you leave out
individual transcription factors during the transcription assay (lanes 9-13). Transcription templates were
preincubated with none, all, or all minus one of the transcription components. The transcription assay was
carried out with all, or all minus one of the transcription components. Use this information and the data below to
answer the questions.
factors present
during:
preincubation
transcription
transcript →→→→
naked
DNA
0 0 all -A -B -D -E -Pol all all all all all
all all all all all all all all -A -B -D -E -Pol
rrrrr
1 2
3
4
DNA assembled into chromatin-
5
6
7
8
9
10 11 12 13
A) Which of the transcription components must be present during the preincubation to keep the template active
during chromatin assembly? Explain your answer.
B) Which of the transcription components form a complex with the template that is stable to chromatin
formation and subsequent chromatin purification? Explain your answer.
C) Which of the transcription components must be added during the assay in order to produce a transcript?
Explain your answer.
Transcribed Image Text:Question 2: You decide to test how the presence or absence of nucleosomes affects transcription using an in vitro transcription assay in which a DNA template featuring a promoter and single reporter gene is transcribed with the addition of specific factors in a test tube. You get robust transcription when you combine template DNA, RNA Polymerase, NTPs, and four general transcription factors - TFIIA, TFIIB, TFIID, and TFIIE. However, if you assemble the template onto purified nucleosomes and purify the chromatin template, then you don't get transcription even when all of the transcription components have been added. You decide to try a slightly different experiment where you first preincubate the template with all the transcription components except NTPs, then assemble the template onto nucleosomes and purify the chromatin template. Now when you add the transcription components and NTPs, transcription proceeds just as well as it does when using the naked DNA template (compare lanes 1 and 3). You conclude that one or more of the transcription factors must bind to the template and keep the promoter accessible. To investigate this phenomena in more detail, you carry out two additional kinds of experiments. In one, you leave out individual transcription factors during the preincubation (lanes 4 – 8). In the second, you leave out individual transcription factors during the transcription assay (lanes 9-13). Transcription templates were preincubated with none, all, or all minus one of the transcription components. The transcription assay was carried out with all, or all minus one of the transcription components. Use this information and the data below to answer the questions. factors present during: preincubation transcription transcript →→→→ naked DNA 0 0 all -A -B -D -E -Pol all all all all all all all all all all all all all -A -B -D -E -Pol rrrrr 1 2 3 4 DNA assembled into chromatin- 5 6 7 8 9 10 11 12 13 A) Which of the transcription components must be present during the preincubation to keep the template active during chromatin assembly? Explain your answer. B) Which of the transcription components form a complex with the template that is stable to chromatin formation and subsequent chromatin purification? Explain your answer. C) Which of the transcription components must be added during the assay in order to produce a transcript? Explain your answer.
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