Transcription Flashcards
Facts about transcription
- the transcription bubble stays the same size throughout
- unidirectional
- only one of the strands is being copied
- the RNA strand is only base paired for a short region of about 9 base pairs
(similarities to replication)
Enzyme: RNA Polymerase
Synthesis: occurs 5’ to 3’
Chemistry: 3’OH attacks phosphate on incoming nucleoside triphosphate
(differences to replication)
- synthesized strand is made out of ribonucleotides rNTPs
- RNA polymerase does not require a primer to initiate synthesis
- RNA polymerase can unwind the duplex DNA on its own
- The RNA product does not remain base-paired to the template DNA
- only one strand is transcribed
- transcription is much less accurate than DNA replication
- during transcription specific genes are selectively transcribed through a highly regulated processes whereas during DNA replication the entire genome must be replicated exactly one time each cell cycle
What are the 5 core subunits in RNA Polymerase?
B B’ a’ a’’ w
What are the similarities between RNA polymerases in prokaryotes and eukaryotes?
The CRAB CLAW configuration with an Mg ion in the middle required for catalysis along with the 5 core subunits.
What is the chemistry of RNA Synthesis?
RNA polymerase has 2 Mg2+ ions. One shields the negative charges on the beta and gamma phosphates and the other shields the negative charges on the alpha phosphate along with the 3’OH.
Upstream means…
toward the 5’ of a given sequence
Downstream means…
toward the 3’ of a given sequence
Direction of Transcription
RNA is always made in the 5’ to 3’ direction
DNA template strand in comparison to RNA
template strand/antisense strand
-complementary and antiparallel to RNA
DNA coding strand in comparison to RNA
coding strand/ sense strand
-same sequence as RNA except it has thymine instead of uracil
Either strand of the gene can be used as a template strand. T/F
True. This depends on where the promotor is. It can be on the template or coding strand. This is different for different genes but for each unique gene, it is the same.
Initiation of Transcription
RNA polymerase finds the promotor sequence and binds to it in the closed complex form which means the polymerase is bound to a closed double stranded DNA helix- this is easily reversible. Then it undergoes isomerization and a transition occurs to the open complex of single stranded DNA which involves a structural change in the polymerase. This is very stable and transcription is guaranteed to happen- essentially irreversible. this transition does not require energy ATP in prokaryotes
The beginning of transcription is actually inefficient because a consistent ~10nt strand needs to be made in order for transition to continue.
Elongation of Transcription
A stable and efficient complex is formed with RNA polymerase and the DNA and RNA is made.
What tells RNA polymerase where to start and stop transcription?
promotor and terminator sequences
______ is required for transcription initiation to bring RNA polymerase specifically to promotor sequences.
The sigma factor
RNA Polymerase Holoenzyme
Bacterial RNA
This is the bacterial core of RNA Polymerase + the initiation factor sigma. RNA polymerase holoenzyme only initiates transcription at promoter sites in vivo due to the presence of sigma
sigma 70 is the predominant sigma factor in E. Coli
Features of a Promoter in Bacteria
Consensus Sequence for sigma70
- UP- element: not always present but when it is, it is better
- -35
- -10 (TATATT)
- both the -35 and -10 are double stranded and can be recognized from either template strands
- they are (-) meaning they are upstream of the transcription start site
How far apart are the preferred sequences of -35 and -10 for the sigma factor promoter?
17 base pairs
Promoter “Strength”
This refers to how many transcripts can be initiated at a given time for a given gene
- Pol must be able to bind efficiently to the promoter
- Promoter must support isomerization
- Pol must be able to escape the promotor
What is the difference between strong and weak promoters?
- strong promoters have sequences that closely match the consensus and are highly expressed– may also contain the UP-element
- weak promoters dont match the consensus as well and are expressed at lower levels
Which two subunits recruit RNA polymerase to the promoter?
sigma and alpha
- alphaCTD binds to the UP element
- alpha NTD contains sigma4 and sigma 2 that bind to -35 and -10 respectively
Recognition of specific promoter elements involves __________insertion into DNA ______ and _______.
Recognition of specific promoter elements involves alpha helix insertion into DNA major groove and H-bonding.
Alternative sigma factors recognize different promoter sequences- provides a form of transcriptional regulation. T/F
True
How is the DNA melted into a single stranded form (open complex) when RNA polymerase binds to it?
It is driven by interactions between sigma70 region 2 and the -10 element. Two bases in the -10 element (non-template strand) flip out and insert into pockets of the sigma2 region. This occurs here because there is ATAT base pairing which only consists of 2 H-bonds.
How many channels does RNA Polymerase have?
- DNA entry
- DNA exit
- product RNA exit
- entry for substrate rNTPs
Transition from Initiation to Elongation
Abortive Initiation
-short
Elongating Complex
- unwinds DNA in the front
- reanneals DNA in the back
- synthesizes RNA
- dissociates RNA from the template
- 2 proofreading functions
**Notice how elongating RNA Polymerase has many jobs, compared to DNA replication
-During elongation, polymerase uses a step mechanism and translocates the distance of one nt after each new rNTP is added. the size of the transcription bubble remains constant.
- 3 step cycle:
1. binding of rNTP
2. catalysis of new phosphodiester bond
3. translocation
What are the two proofreading functions for RNA Polymerase?
*these occur at the same active site so there is no movement of the transcript
- Pyrophosphorolytic Editing: reverses the process by re-entering the PPi that was just removed
- Hydrolytic Editing: H20 is used to removal bases containing the mismatch
Termination of Transcription in Prokaryotes
Terminators: sequences in the DNA at the end of genes that trigger the elongating polymerase to dissociate from the DNA and release the synthesized RNA strand
-2 forms: Rho-dependent and Rho-independent
Rho transcription Termination Factor
- Hexameric ring-shaped protein with 6 identical subunits
- ATPase activity
- A member of the family of ATP-dependent hexametric helicases
Rho-dependent termination
rut sites: rho utilization site- RNA elements of approx 40 nuts; initial rho binding site (upstream of the actual terminator)
*sequence specific binding
Rho-independent termination
Intrinsic Terminators
Consist of:
1. a short inverted repeat of 20 nuts followed by…
2. a stretch of 8 A:U bps
(A- template dna and U in RNA transcript)
*A:U is the weakest base pair there is!
