Transcription Flashcards
What is transcription?
Using DNA as a template to synthesize RNA
Why do we need to say that a gene is a sequence of nucleic acids that encodes a functional product, not a sequence of DNA that encodes a protein?
RNA viruses exist, and they have RNA that encodes a functional product. There are also genes that encode tRNA and rRNA (and other types of RNA), so the end product isn’t always a protein
What is the primary enzyme used in transcription?
RNA polymerase
What is used as a template for transcription?
DNA
What are the subunits being assembled during transcription?
NTPs
What are the 3 key steps in transcription?
Initiation, elongation, termination
Which direction is the coding strand of DNA read during transcription? How does the mRNA look compared to this strand?
5’ to 3’. The mRNA produced is identical to the coding strand with all the thymines replaced with uracil
Which direction is the template strand of DNA read during transcription? How does the mRNA look compared to this strand?
3’ to 5’. The mRNA produced is complementary to the template strand since this is the strand that RNA polymerase reads
What defines the beginning of transcription?
The promotor, where RNA polymerase binds
What is the +1 nucleotide?
Where transcription is initiated and the RNA transcript begins to be assembled
What defines the end of transcription?
The terminator
What defines the promotor regions in prokaryotes?
The two consensus sequences at -10 and -35 with high affinity for the sigma subunit of RNA polymerase
Why is the sigma subunit important? What happens to RNA polymerase without it?
The sigma subunit is what physically binds to the two consensus sequences. Without it, RNA polymerase is able to elongate the strand of mRNA but loses the specificity of binding to the promotor and will be able to bind to anything
What is RNA polymerase called when the sigma subunit is present?
The RNA polymerase holoenzyme
What are the 4 steps of transcription initiation in prokaryotes?
- The RNA polymerase holoenzyme binds to DNA
- Binds tighter at the promotor
- Tight binding promotes local melting of the DNA into single strands
- Transcription begins and RNA polymerase continues to unwind the double helix as it continues to transcribe
Does RNA polymerase need a primer?
No, it can synthesize de novo
What is happening inside of RNA polymerase during elongation in prokaryotes? What happens to the DNA after it is transcribed? What is happening to the mRNA being synthesized?
Only a short stretch of synthesized mRNA remains base paired to to DNA inside RNA polymerase. The DNA reanneals as a double helix after it leaves RNA polymerase and the synthesized mRNA protrudes out of the enzyme
What is processive transcription? What contributes to RNA polymerase being able to processively transcribe?
Processive transcription is saying that RNA polymerase is good at continuously catalyzing the reaction, so it won’t fall off until it is supposed to. Tight binding to DNA, RNA, and the DNA/RNA hybrid helix allows transcription to be processive
How does RNA polymerase proofread? What will make it stop transcribing to proofread?
RNA polymerase will occasionally stop and backtrack about 5 base pairs to proofread. If it finds a mismatch, it uses endonucleolytic cleavage to cut off the strand and then resynthesizes it. If no mismatch is found, it goes back to where it was and keeps transcribing. A weak DNA/RNA hybrid helix will cause it to stop from a lack of hydrogen bonds
What happens to the DNA/RNA hybrid helix during transcription termination?
It gets disrupted, destabilized and unwound
What is Rho?
A helicase involved in transcription termination in prokaryotes
What happens in Rho dependent termination?
- Rho binds to the rut site in the RNA transcript
- It follows RNA polymerase along the transcript
- When RNA polymerase transcribes the terminator sequence, it will pause and Rho can catch up
- Rho unwinds the RNA/DNA hybrid helix
How does the terminator sequence cause RNA polymerase to pause in Rho dependent termination?
The terminator sequence forms a stemloop, which physically pulls on the RNA still in RNA polymerase and weakens the RNA/DNA hybrid helix. The weakened duplex from the stemloop causes the pause
How does intrinsic termination work in prokaryotes?
A GC rich sequence gets transcribed which forms a stemloop, then a polyU sequence is transcribed. These two things destabilize the DNA/RNA hybrid helix enough for RNA polymerase to stop and fall off
How is RNA polymerase different between eukaryotes and prokaryotes?
Eukaryotes have different versions of RNA polymerase that create different kinds of RNA, while prokaryotes only have the one. The core of RNA polymerase is conserved from prokaryotes to eukaryotes, but eukaryotes have no homolog for the sigma subunit
Can eukaryotic RNA polymerase initiate transcription on its own?
No. It requires general and specific transcription factors to initiate transcription
What is the difference between general and specific transcription factors?
General transcription factors are always required for transcription initiation at every gene in eukaryotes. Specific transcription factors regulate transcription at certain genes
What do the general transcription factors do to initiate transcription in eukaryotes?
They bind to the promotor sequences and recruit RNA polymerase II. They also contain helicase activity that promote local melting
What are the steps in transcription initiation in eukaryotes?
- The general transcription factor recognize and bind to the promotor sequence
- They recruit RNA polymerase
- One of the general transcription factors phosphorylates the tail of RNA polymerase and recruits the elongation factors
- DNA melting occurs and transcription begins
Are the promotor sequences exactly the same in all eukaryotic promotors?
No, there are lots of possible sequences, and each promotor will have a different combination. Each sequence is bound to by one specific general transcription factor
What is the TATA box? What binds to it?
A promotor sequence that is commonly found in eukaryotic promotors. TBP binds to it - TATA binding protein
Are the general transcription factors involved in elongation in eukaryotes?
No, elongation factors are there instead. The general transcription factors are only for initiation and will stay bound at the promotor
What do the elongation factors do?
RNA processing: splicing, capping, polyadenylation. One also does endonucleolytic cleavage after the terminator is transcribed
What happens when the terminator sequence is transcribed in eukaryotes?
- An elongation factor cuts the mRNA right after the terminator sequence
- RNA polymerase continues to transcribe
- Rat1 (an exonuclease) degrades the excess RNA until it reaches the RNA/DNA hybrid helix
- The hybrid helix is disrupted and RNA polymerase falls off
