Lecture 8: Transcription Flashcards
(28 cards)
What is the difference between ribose and deoxyribose?
Ribose sugar has an oxygen on the second carbon and deoxyribose does not
What polymerase successively adds ribonucleotides to a growing mRNA transcript?
RNA Polymerase II
What nucleotide is present in RNA and not DNA? What does it base pair with?
Uracil. It base pairs with Adenine
In what direction is the mRNA transcript synthesized?
5’ to 3’ (left to right)
What is the substrate used by RNA polymerase in the process of transcription?
Ribonucleotide Triphosphate
What is the gene nucleotide that RNA Polymerase being at for transcription called? What is considered upstream an downstream relative to this location?
The gene nucleotide it starts at is designated at ‘+1’. The nucleotides after this (towards 3’ direction) is considered downstream and allocated positive numbers. The nucleotides above the +1 position (towards 5’) are considered upstream and are allocated negative numbers. Note: RNA polymerase travels downstream when synthesizing the mRNA transcript
In terms of the gene structure, what is in the upstream and downstream region?
Upstream = promoter sequences and regulatory sequences
Downstream = coding region
Note: site of transcription = +1
Define the template and non-template strand of DNA when it comes to transcription
Template strand: strand that is being transcribed
Non-template strand: complement to the template strand, same as mRNA transcript except it has U instead of T
What happens in each of the three stages of transcription?
Initiation:
- Polymerase binds to the promoter sequence (in eukaryotic RNA polymerases they have general transcription factors that identify promoters and initiate transcription)
- Locally denatures DNA forming a transcription bubble and catalyzes the first phosphodiester linkage in mRNA transcript
- Promoter sequence is upstream
Elongation:
- Polymerase advances 3’ to 5’ down template strand (synthesizing mRNA in 5’ to 3’) denaturing the DNA and polymerizing the RNA
Termination:
- Polymerase recognizes a stop site and releases the primary mRNA transcript and dissociated from DNA
- Specific sequence in the template DNA signals the bound RNA polymerase to terminate transcription
What its the role of the sigma factor in transcription?
To recognize specific DNA sequences as promoters and recruit RNA polymerase. After transcription is initiated the sigma factor is released
What is a sigma factor? What sequences do s70 and s54 recognize?
A transcription initiation factor that can chemically recognize sequences as promoters – can regulate whole families of genes
s70:
- recognizes promoter sequences with TTGACA—TATAAT
s54:
- recognizes promoters of genes involved in nitrogen metabolism
What are the three stages of transcription?
Initiation, elongation and termination
What is the difference in how genes are organized and in eukaryotes vs. prokaryotes?
Prokaryotes:
- Have very few non-coding gaps (introns) of DNA in prokaryotic genome
- Genes with a common function are arranged physically together in a line and transcribed together on a single mRNA – when it comes to translation, to get different proteins they just have different start sites on the mRNA transcript (reference image)
Eukaryotes:
- Genes with common function are not restricted to be physically close to each other for regulation
- Different transcripts can give rise to different proteins
What is the difference in transcripts between in eukaryotes vs. prokaryotes?
- In prokaryotes, mRNAs are directly transcribed from DNA.
- In eukaryotes, transcripts must go through several processing steps before becoming mRNAs.
What is the structure of Eukaryote RNA Polymerase
Contains two large subunits and 10–14 smaller subunits, some of which are common between two or all three of the polymerases, whereas others are specific.
Fill in the black: RNA Pol II ___ subunit ___ contains an essential ____
- Largest
- RPB1
- C-terminal domain (CTD)
Is RNA Polymerase II highly conserved in all eukaryotes?
Yes
What is the chemical composition of the CTD on RPB1?
In mammals, the CTD is made up of 52 nearly identical repeats of Tyr- Ser-Pro-Thr-Ser-Pro-Ser – phosphorylation of aa on this tail by transcription factors influence transcription
What does the phosphorylation of the CTD on RPB1 mean in terms of transcription?
- RNA polymerase molecules that initiate transcription have an unphosphorylated CTD.
- RNA polymerase molecules that are actively transcribing have phosphorylated CTD
What is the role of the clamp domain of RPB1 during transcription?
- It is open when downstream DNA is inserted into this region of the polymerase.
- It swings shut when the enzyme is in elongation mode — anchors polymerase onto the downstream double-stranded DNA. Shut position makes enzyme very processive
How does free RNA Pol II differ from a transcribing one (hint: think of the clamp domain)?
Transcribing RNA polymerase II:
- It differs from free RNA polymerase II mainly in the position of a clamp domain in the RPB1 subunit
- Clamp domain swings over the cleft between the jaws of the polymerase during formation of the transcribing complex, trapping the template DNA strand and transcript (ref. image in notes)
- Mg2+ ion participates in catalysis of phosphodiester bond formation – critical component of that process
List the RNAs transcribed by each of the the RNA polymerases. If possible state the function of these RNAs
RNA Pol I
- Pre-rRNA: Ribosome components, protein synthesis – making rRNA
RNA Pol II
- mRNA: Encodes protein
- snRNAs: RNA splicing
- siRNAs: Chromatin-mediated repression, translation control
- miRNAs: Chromatin-mediated repression, translation control
RNA Pol III
- tRNAs: Protein synthesis
- snRNA U6:RNA splicing
What is the role of the promoter element
Specify where polymerase initiates transcription of an RNA complementary to the template strand of the gene DNA
What can single base change in the TATA box lead to
can decrease gene transcription
