Transcription l Flashcards
The biological role of transcription:
Transcription is the synthesis of an RNA molecule from a DNA template by the enzyme DNA polymerase
In eukaryote the mRNA undergoes few modifications before it leaves the nucleus to be translated
to synthesis protein by ribosome.
Is RNA molecule a single strand? How does it behave? Why?
RNA is a single strand, but it does not behave like a piece of string. Intramolecular base pairing
defines 3D shape - provides strength
What is coding strand? And what is the template strand? What determines which one is which?
- Only one of the two strands of the DNA serve as the template for transcription.
- Which strand it is – top or bottom, will depend on the gene
Why temporary base pairing occurs between mRNA and DNA?
Yes, DNA/RNA hybrid forms , temporary base pair binding, the base pairing is what determines the information which should be transcribed into mRNA
what is the Relationship between template strand, coding strand and synthesized RNA ?
The messenger RNA must be made from
one strand of DNA called the template strand.(complementary)
The other strand, called the coding strand,
matches the messenger RNA in sequence except
for its use of uracil in place of thymine.
What type of a chemical bond does RNA polymerase form?
Phosphodiester bond
Where does the energy for RNA synthesis come from?
The energy released through the hydrolysis of a high energy phosphate bond in ribonucleotide
triphosphates is used to power strand separation and translocation along DNA during transcription
(nucleophilic reaction)
RNA polymerase generates a ……
RNA polymerase generates a transcription bubble, which separates the two strands of the DNA helix.
This is done by breaking the hydrogen bonds between complementary DNA nucleotides. RNA
polymerase adds RNA nucleotides (which are complementary to the nucleotides of one DNA strand).
Is it true that in both prokaryotes and eukaryotes there is only one type of RNA polymerase? why?
Why not?
One RNA polymerase synthesizes all different types of RNA
Transcription in eukaryotes: Three separate RNA polymerases: RNA Polymerase I; RNA
Polymerase II; RNA Polymerase III
Many molecules of RNA polymerase simultaneously transcribe one gene
the growing RNA is carried along with RNA polymerase as it moves
the longer RNA polymerases transcribe= the longer the RNA molecule carried with is
How does RNA polymerase ‘decide’ which strand to use and where to start transcription?
Finding the promotor: A promoter is a sequence of DNA needed to turn a gene on or off. The
process of transcription is initiated at the promoter. Usually found near the beginning of a
gene, the promoter has a binding site for the enzyme used to make a messenger RNA
(mRNA) molecule.
site +1 and 5’ UTR region
The site +1 denotes the first DNA base to be transcribed (the initiation site).
The transcription initiation site +1 is usually well upstream of the start codon ATG. The intervening
part is referred to as the 5’UTR – 5’ untranslated region.
Where is the protein coding strand? after the promoter, next to the promotor ? and what is the start codon ?
Downstream of the promoter is the protein coding sequence The protein coding sequence of the gene begins with the start codon ATG.
Stop codon
end with a stop codon (usually with a double stop codon TAA TAA )
-10 and -35 position stream
prokaryotes
In prokaryotes, the promoter consists of two strongly conserved short sequences at -10 and -35 positions upstream from the transcription start site.
- sequence at -10 is called the Pribnow box, six nucleotides TATAAT. absolutely essential to start transcription in prokaryotes.
- sequence at -35 (the -35 element). six nucleotides TTGACA. Its presence allows a very high transcription rate
- important for recognition by RNA polymerase.
Transcription initiation steps prokaryotes
Step 1: the RNA polymerase holoenzyme (core enzyme plus sigma-factor) assembles and then locates
the promoter
forms of prokaryotic RNA polymerase:
- core enzyme (composed of 5 subunits),
- holoenzyme (core plus the sigma factor subunit)
- Different types of sigma factor for different RNA synthesis
- Sigma factors dissociate once the transcription
bubble is formed
Function of sigma factor
helps to locate promotor- identifies the region -10 and -35
–one side of the sigma factor binds to the -35 and -10 regions of the promoter on the DNA, and the
other side forms extensive interactions with the other subunits.
- Different types of sigma factor for different RNA synthesis
how sigma factor determine which gene to transcript?
DNA is unwound around the -10 region and one strand of it is fed into the channel in RNA polymerase that goes all the way through the molecule, and that incorporates the active site marked by the position of the Mg+ ion.
therefore the sigma factor that determines which genes are transcribed.
Difference between enhancer and promotor
An enhancer is a sequence of DNA that functions to enhance transcription. A promoter is a sequence of DNA that initiates the process of transcription.
A promoter has to be close to the gene that is being
transcribed while an enhancer does not need to be
close to the gene of interest.
Transcription elongation-
The transcription elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core RNA polymerase enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction at a rate of
approximately 40 nucleotides per second
Transcription termination- Prokaryotes
Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA
- -Intrinsic terminators (do not require any other factors) -more common
- Rho dependent terminator sequences - less common
Intrinsic terminator sequence
The RNA transcript of the intrinsic terminator sequence has a GC-rich inverse repeat sequence followed by a string of Us.
Intrinsic termination
• The two inverse repeat sequences are complementary to each other. RNA anneals onto
itself in this region, forming a very stable GC-rich hairpin loop.
• When the string of Us string is synthesized in the transcription bubble, it complements the string
of As on the template DNA. The two are held together by weak hydrogen bonds, this bonding is
unstable
• When RNA polymerase encounters an unstable strand pairing in the transcription bubble, it normally backtracks to stabilize it. But in this particular case the hairpin loop creates a ‘roadblock’ resulting in a release of RNA and dissociation of RNA polymerase from the DNAtemplate.
Rho (ρ)-dependent chain termination - prokaryotes
requires the help of the Rho factor.
• The termination site in this case is preceded by an
upstream Rut sequence (Rho utilization).
• As RNA polymerase transcribes the Rut sequence, it
creates the Rho binding site on RNA.
• Rho binds to RNA
• As RNA polymerase moves further along the DNA, the Rho factor then moves along the newly synthesized RNA strand, trying to catch up with RNApolymerase. Once RNA polymerase reaches the terminator sequence, it pauses, and Rho finally catches up. Rho isan ATP dependent motor that uses the energy of ATP hydrolysis to unwind the DNA-RNA
transcription bubble resulting in the release of RNA and all protein components and termination of
transcription.
