Lecture 4: Transcription & Translation Flashcards
What are the basic requirements of transcription?
• Ribo-nucleoside triphosphates (NTPs) to create the new strand
• A template to copy
• An enzyme to do the polymerisation: RNA Polymerase
• An energy source
What are two common toxic inhibitors of transcription and translation? How do they work?
Alpha-aminitin and Ricin.
• Alpha-aminitin – Amatoxin that inhibits RNA polymerase II, causing protein deficit and ultimately cell death.
• Ricin – Lectin (protein that binds to carbohydrates) is a poison found naturally from castor beans. A-chain inactivates ribosomes by hydrolysing the N-glycosidic bond of an adenosine residue; this inhibits protein synthesis by blocking the binding of elongation factors.
What are some helpful transcription and translation inhibitors? What do they do?
Rifampicin and Chloramphenicol.
• Rifampicin – Produces bactericidal antimicrobial activity by inhibiting DNA-dependent RNA polymerase (RNA-P) either by sterically blocking the path of the elongating RNA at the 5’ end or by decreasing the affinity of the RNAP for short RNA transcripts.
• Chloramphenicol – Antibiotic that works by inhibiting protein synthesis by binding to the 50S ribosomal subunit and directly preventing the formation of bacterial protein. Blocks prokaryotic translations.
What are the challenges in transcription?
Since not all of a genome is transcribed, the transcriptional machinery must find the correct regions of the genome to transcribe.
How is the challenge of DNA transcription resolved?
A gene contains information that directs the transcriptional machinery to the correct place to start and stop transcription. —>
–> Promoter and termination regions.
What is RNA Polymerase (RNAP)?
RNA Polymerase is a multi-subunit (multi protein components) enzyme that generates RNA strands (transcripts) using a DNA template and ribonucleotides.
Features and purpose of DNA promoter region.
Contains specific sequences to recruit RNA polymerase and regulatory proteins (activators/repressors).
Promoters contain DNA sequences that are recognised by RNA polymerase.
What are RNA transcripts?
RNA strands that are synthesised using RNAP.
Describe the transcription termination region.
It contains specific sequences to indicate where transcription should stop.
Characteristic of most prokaryotic promoters.
They contain conserved consensus sequences at -10 and -35.
What are conserved consensus sequences?
By consensus sequences, short stretches of nucleotides that occur multiple times in conserved sequences.
~ Calculated sequence of most frequent residues (nucleotide or amino acid) found at each position in a sequence alignment.
~A sequence of DNA having similar structure and function in different organisms.
By conserved sequences, base sequences in a DNA molecule that have remained essentially unchanged throughout evolution.
Common nucleotides at each position that were preserved through the evolution in species (?)
How does RNAP bind to C.C. sequences in prokaryotic promoters?
RNAP binds to these sequences via the sigma subunit (factor).
What is the function of sigma subunit?
It conveys promoter specificity to RNA polymerase; that is, it is responsible for telling RNA polymerase where to bind.
Because there are number of different sigma subunits that bind to different promoters and therefore assist in turning genes on and off as conditions change.
What is -35 region responsible for?
Rate of transcription.
What is -10 region also known as? What is its function?
-10 region is also known as Pribnow box, its role is initiation of transcription.
Where is the first RNA nucleotide transcribed?
In the +1 Transcriptional start site.
AT-rich nature of promoter sequences aids what?
Separation of the DNA strands.
In what direction does the RNAP move along the template strand?
3’ to 5’ direction.
In what direction does the transcription bubble go in?
3’ to 5’.
What is transcription bubble?
It is formed when the RNAP enzyme binds to a promoter and causes two DNA strands to detach.
It presents a region of unpaired DNA, where a short stretch of nucleotides are exposed on each strand of the double helix.
Non-template strand is also called what?
Partner or Coding strand.
Why is the non-template strand called coding strand?
Because its sequence will be the same as that of the new RNA molecule, that would end up coding for proteins.
Characteristics of the mechanism of transcription.
• Relies on complementary base-pairing.
• Nucleotides are added to the 3’ end of the RNA chain.
How does the elongation process occur in transcription?
Addition of ribonucleotides to the 3’ end of the growing script. Nucleophilic attack of -OH.
Hydrolysis of the pyrophosphate releases energy to drive transcription.