Transcription Flashcards
What kind of template is required for RNA synthesis?
A DNA template
What is RNA synthesized by?
By RNA polymerase
What direction is RNA synthesized?
5’ to 3’
Is a primer needed for RNA synthesis?
Nope
Do RNA polymerases proofread?
No, there are lots of mRNA copies, so occasional errors ok.
How many different RNA polymerases does E. Coli use for all transcript synthesis?
rRNA, tRNA and mRNA all use the same RNA polymerase
What are the 3 stages of RNA synthesis?
Initiation
Elongation
Termination
Where is transcription initiated?
At promoter regions on the template/coding strand
Which strand do we refer to when talking about the initiation site?
The sense strand even though the template strand is transcribed
Where is the site of transcription located in a gene?
5’ upstream of position of translational initiation codon of the sense strand
What is the transcriptional start site?
The first base on template transcribed/sense strand copied
Can promoter efficiency be increased or decreased?
Yes, by introducing mutations
How does RNA polymerase initially form a closed complex to DNA?
The holo enzyme binds weakly to the DNA, slides along the -35 site then binds closely.
What type of nt is almost always the first brought into the active site?
Purine
What is the sigma subunits required for?
Ensures specific, efficient recognition of the promoter
Once several bases incorporated the subunits is lost
What do different types of sigma subunits allow?
Different promoters to be used in transcription
When is the first phosphodiester bond created?
When the next nucleotide aligns with the template
How fast does RNA polymerase add bases?
About 50 bases/sec
What happens in the winding of DNA during elongation?
The DNA helix is unwound in front of the enzyme and rewound behind of the enzyme
What does rewinding do with RNA?
Displaces the growing RNA strand
Where does termination occur?
Some distance 3’ downstream of the translation termination codon
In termination, why does RNA polymerase dissociate?
Because once the nascent RNA chain is released the polymerase is no longer able to bind
What does DNA do when it goes through termination?
The helix rewinds
What are the two mechanisms of transcriptional termination?
Rho dependent
Rho independent
What happens in Rho dependent termination?
The Rho protein binds to poorly characterized sequences in 3’ region of transcript and uses energy from ATP to break hydrogen bonds holding transcript to template.
What is Rho independent termination driven by?
Secondary structure formation of RNA transcript and thermodynamics
What happens in Rho independent termination?
Region of transcription folds into a U rich hairpin which reduces base pairing to just the template which has poor hydrogen bonding to template so favors DNA rewinding
What does RNA polymerase I transcribe and where can it be found?
rRNA in the nucleolus
What does RNA polymerase II transcribe and where can it be found?
mRNA and in the nucleus
What does RNA polymerase III transcribe and where can it be found?
tRNA and 5S rRNA in the nucleus
How sensitive is RNA polymerase I to alpha-amanitin?
Not
How sensitive is RNA polymerase II to alpha-amanitin?
Super
How sensitive is RNA polymerase III to alpha-amanitin?
Pretty sensitive
Describe the core enzyme of RNA polymerase II?
It is a multisubunit protein and requires numerous additional proteins for efficient initiation of transcription.
Where is the mRNA promoter TATA Box located?
25 bases upstream of +1 transcription start site
What is required for efficient transcription to begin at the TATA box?
RNA polymerase II as well as numerous DNA sites(cis-acting elements, enhancers) and associated proteins that bind to these sites (transcription factors).
What is the function of Pol II?
Catalyzes RNA synthesis
What is the function of TBP?
Recognizes TATA box
What is the function of TFIIA?
Stabilizes TFIIB and TBP binding to promoter
What is the function of TFIIB?
Binds TBP; recruits pol II-TFIIF complex
What is the function of TFIIE?
Recruits TFIIH; has ATPase and helicase activities
What is the function of TFIIF?
Binds pol II; binds TFIIB and prevents nonspecific binding of pol II
What is the function of TFIIH?
Unwinds DNA at promoter; phosphorylation pol II within CTD
What is a newly synthesized RNA molecule called?
Primary transcript
Does mRNA need to go through processing modification?
In prokaryotes no
In eukaryotes yes
All other transcripts other than mRNA in prokaryotes must go through processing.
What sort of modifications do transcripts other than mRNA in prokaryotes go through?
Base modifications (tRNA) Cleavage of large primary transcript into functional fragments (rRNA)
What happens in base modifications?
Bases are chemically modified
Can primary transcripts be cleaved to produce functional fragments?
Yes they can!
What are nucleoli?
Ribosomal factories
What happens in the nucleolus?
rRNA and ribosomal proteins are arranged into large and small ribosomal subunits and shipped to the cytoplasm
What happens to mRNA in eukaryotes when processed?
Introns are spliced, a 5’ cap and 3’ poly A tail are added
What is mRNA splicing mediated by?
Small nuclear RNAs (snRNAs) called U1 to U6 and formation of a spliceosome complex
How is the lariat structure formed in mRNA splicing?
OH groups serve as nucleophiles to form the structure then join the donor and acceptor junction.
When are mRNAs edited before translation?
When the mRNA sequence does not precisely match complementary DNA sequence.
What sort of editing does RNA undergo?
Addition, deletion and alteration of nucleotides
When does addition/deletion of nucleotides occur?
In mitochondrial transcripts
What does alteration usually involve?
Enzymatic deamination of A or C to form inosine (interpreted as g by translational machinery) or uridine.
Why is protein produced in the intestine shorter than protein produced in the liver?
CAA (gln) is converted to UAA (stop)
What are 4 inhibitors of transcription?
Rifamycin
Alpha-amanitin
Actinomycin D
Acridine
