Central Dogma Lecture 11 Transcription I Flashcards
(33 cards)
What are the four major classes of RNA? What do they do?
-mRNA (messenger RNA):
-Encodes information for synthesis of proteins
-rRNA (ribosomal RNA):
-Components of ribosomes (translation machinery)
-Comprises 90% of a cell’s total RNA
-tRNA (transfer RNA)
-Used to carry amino acids to mRNA-ribosome complex during translation
-ncRNA (noncoding RNA):
-Diverse group with catalytic, structural, and/or regulatory functions
~76% of the human genome is transcribed; most products are ncRNAs
What polymerase is used for RNA in bacteria
-RNA polymerase (RNAP)
-DNA-directed synthesis of RNA
-In bacteria, one type of RNA polymerase synthesizes all RNA, except primers used in DNA replication
What makes up the RNAP holoenzyme?
-Core + sigma factor (𝜎)
- 𝜎 factor dissociates once RNA synthesis has been initiated
What catalyzes the actual polymerization?
-The RNAP core
-Made up of α, β, β’, and ω subunits
What do sigma factors do?
-Sigma factors allow RNAP to slide rapidly on DNA in search of promoter
-Holoenzyme (core + sigma) binds loosely to duplex DNA
-Different sigma factors direct transcription of different genes
-Sigma factor specifies the promoter that RNAP holoenzyme forms a stable complex with
-All sequence specific contacts are mediated by the sigma factor
Promoter basics in bacteria
-RNA synthesis is initialted at specific DNA sequences called promoters
-Upstream and have negative numbers
-Usually A or G
Where do RNAPs bind promoters in bacteria?
-RNAP binds tightly to promoters at 2 highly conserved regions
-Centered at -10 position
-Centered at -35 position
-Sequence in between is unimportant, but length is critical (16-19bp)
What does transcription rate depend on in bacteria?
Rate of promoter forming a stable initiation complex with RNAP enzyme
What happens with sigma factor when transcription has been initiated?
-Sigma is jettisoned
-Core by itself binds tightly to duplex DNA
How are bases to be transcribed for RNA numbered?
-First base of RNA that is transcribed is marked as +1
-No 0
What is the antisense strand?
-The template strand (or noncoding)
-Strand that is transcribed
What is the sense strand?
-Coding strand (or non-template strand)
-Has same sequence as the transcribed RNA
-Promoter is on the DNA’s coding/non-template/sense strand
Which strand is the promoter on in DNA?
The coding/sense strand
Where does RNAP holoenzyme bind promoter?
-On the -10 and -35 regions on the same side of DNA helix as initiation site
-Binds only to one face of the promoter B-form helix
Formation of the transcription bubble
-Unclear how exactly this “invasion” happens
-Binding of RNAP holoenzyme “melts” DNA from center of -10 region to just past the initiation site
-Promoter efficiency decreases with # of G-C pairs in -10 region
What occurs with the bubble during processive transcription?
Eight bases of RNA transcript are hybridized with DNA in the bubble
What is the shape of the RNAP core structure?
-Shape of a crab claw whose two pincers are formed by the β and β’ subunits
-In main channel (between pincers), DNA pairs with incoming NTP
-RNA exits from between β and β’ subunits (the RNA exit channel
How does chain initiation occur?
-The 5’ most base of RNA is almost always A or sometimes G
-Initiating reaction is simply the coupling of two nucleotide triphosphates
-Bacterial RNAs have 5′-triphosphate group (incorporate γ-32P ATP)
-Basically initiation is started by adding two phosphates to the first base?
Abortive initiation
RNAP sometimes fails to escape the promoter and releases newly synthesized RNA after ~10 nt
What happens in a successful initiation?
-RNAP commences processive transcription of the template and jettisons sigma factor
-Transition from initiation complex to elongation complex
-Sigma factor can then join another core to form a new initiation complex
What is the chemical mechanism of transcription?
-RNA chain elongation occurs 5’ to 3’ (same as DNA)
-Amino acid sequence “NADFDGD” forms the RNA polymerase active site
-The three D (Asp) residues are complexed to the Mg2+ ions
Supercoiling in Transcription DNA
-Transcription bubble travels along DNA with RNAP in straight line
-DNA’s helical turns are pushed ahead, causing positive (left-handed) supercoiling
-DNA behind the bubble is underwound, causing negative (right-handed) supercoiling
-Topoisomerases deal with supercoiling
What is the rate of transcription and why is it processive?
-20-70nt/ second
-RNAP acts as its own clamp by binding tightly to DNA-RNA complex
How often does transcription happen?
-For RNAs needed in large quantities, synthesis is initiated as often as sterically possible, about once per second
-Once an RNAP has begun elongating, another can follow
