Case Study 7- Mechanisms Of Action corrections
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Having brushed up on the basics of Prokaryotic Genome Organization and replication, you thought that you were ready to start conducting lab work. However, looking over the planned project, you realize that while you’ve spent all this time focusing on the bacterial genome, gene expression (transcription and translation) is what really matters when it comes to what a cell is actually
doing. While Dr. Saxe thinks you’re ready, you decide to cement your knowledge of transcription and translation before you get into the lab. Basics of Transcription
1.
Where in the eukaryotic cell does transcription take place?
Would be within the nucleus.
2.
Where in prokaryotic the cell does transcription take place?
Will take place in the cytoplasm.
3.
The image above shows a cycle that occurs during transcription initiation and elongation.
a.
What is represented by the blue circle?
The Sigma Factor
b.
What is represented by the red oval?
The RNA polymerase
c.
What is represented by the red line?
The messenger RNA.
d.
Describe what is happening in this image.
What is seen in this picture is the beginning of transcription in a prokaryotic cell. The sigma factor reacts with the RNA polymerase to create a holoenzyme RNA. This is bonded to the promoter region of DNA which starts transcription. Modified from ASM: Collection of Class Activities
-10 Region
-35 Region
Components of a Gene to be Transcribed.
Shown below is a nucleotide sequence of a fragment of double-stranded DNA from E. coli.
5'..TCTACGCATCTAGCGGGCTCTTGACA(17bases)TATAATGCTCAATTGTCGCTAAGGAGGTGCTTGATGATCTGGCGAC
3'..AGATGCGTAGATCGCCCGAGAACTGT(17bases)ATATTACGAGTTAACAGCGATTCCTCCACGAACTACTAGACCGCTG
Assume that the E. coli consensus promoter is:
5'...TTGACA...(any 17
bases)...TATAAT...3'
3'...AACTGT...(any 17
bases)...ATATTA...5'
1.
Overall, what is the purpose of transcription?
To create RNA from DNA.
2.
Where is the consensus promoter sequence found – DNA or RNA? DNA
3.
In which strand(s) of DNA is the promoter sequence found – template/non-coding, coding/non-template, or both?
BOTH strands of DNA contain the promoter region.
4.
How many different variations of promoter sequence are found in a cell’s genome – One
or multiple?
Multiple
5.
What is meant by the “-35” and “-10” regions of the consensus promoter sequence?
The upstream portion of the promoter, which indicates the start of transcription.
6.
What would happen if there was a mutation in the consensus promoter sequence that rendered it completely unusable?
Sigma factor could NOT work with RNA polymerase AND transcription would not happen.
Note: Many mutations from the “Wild Type” sequence don’t render it unusable – sometimes they can even increase the functionality! However, for the purposes of our class, we often assume zero functionality. 7.
What protein recognizes and binds to the promoter region?
Sigma Factor
Modified from ASM: Collection of Class Activities
Modified from ASM: Collection of Class Activities
-10 Region
-35 Region
8.
What enzyme carries out transcription of DNA (i.e., makes the mRNA molecule)?
RNA polymerase
9.
In which direction does transcription take place (left or right on the diagram above)? What determines the direction of transcription?
Left to Right
The lower DNA strand is the Template/Non-Coding strand. Only the RNA that matches the coding strand is produced, meaning the templates strand must be read from.
Because the newly transcribed strand is antiparallel to its template. The newly made RNA will have its’ 5’-end on the left and be synthesized by adding new nucleotides to the 3’- end on the right.
o
Like DNA polymerase, RNA polymerase can only add to the 3’-OH end of the transcript.
10. Which DNA strand will be actively transcribed from (i.e., is the template)? What determines which DNA strand gets copied?
The coding strand.
o
The bottom strand. Recognition of the promoter region by the RNA polymerase +
sigma factor will essentially “ point the RNAp in the right direction” by aligning based on the -35 and -10 regions.
Practicing Transcription
Shown below is a nucleotide sequence of a fragment of double-stranded DNA from E. coli.
5'..TCTACGCATCTAGCGGGCTCTTGACA(17bases)TATAATGCTCAATTGTCGCTAAGGAGGTGCTTGATGATCTGGCGAC
3'..AGATGCGTAGATCGCCCGAGAACTGT(17bases)ATATTACGAGTTAACAGCGATTCCTCCACGAACTACTAGACCGCTG
Assume that the E. coli consensus promoter is:
5'...TTGACA...(any 17
bases)...TATAAT...3'
3'...AACTGT...(any 17
bases)...ATATTA...5'
11. Is the start of transcription a precise location?
There is no definitive start location.
Modified from ASM: Collection of Class Activities
12. Write out the mRNA that will be transcribed from this gene (same as on the first page, just copied here for your convenience), include 5' and 3' ends.
5’- AUUGUCGCUAAGGAGGUGCUUGAUGAUCUGGCGAC-‘3
Hint: The middle of the -10 region of the promoter (TATAAT) occurs approximately 10 bases upstream from the actual start of transcription. Count about 10 bases to the right (downstream) to find the first base that is transcribed.
13. The end of this mRNA molecule is not depicted but could be accomplished by one of two mechanisms. Briefly describe each of these two mechanisms:
Rho dependent termination
Rho binds to RNA and kicks out RNA polymerase from DNA.
Rho independent termination
RNA creates a hairpin loop which creates a series of UUUUUU. Forcing RNA polymerase to fall off.
14. Does the mRNA in prokaryotic cells get processed prior to translation?
No it does not.
15. What does this enable in prokaryotic cells that is not found in Eukaryotic cells?
Translation
Coupled transcription-translation.
16. What happens to mRNA after termination?
It falls off and gets recycled.
It gets degraded by RNAases
Modified from ASM: Collection of Class Activities
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