Transcription Flashcards
What synthesizes RNA?
RNA polymerases
What things are necessary for RNA synthesis?
DNA template
Ribonucleotide triphosphates
Mg2+ cofactor
What are the two complementary base pairs in RNA?
CG
AU
Is a primer needed to initiate RNA synthesis?
No
Why is error in RNA polymerization higher than in DNA polymerization?
They don’t proofread - there are a lot of mRNA copies, so the occasional error doesn’t matter
How many RNA polymerases doe E. coli use for transcript synthesis?
1
What does primase do in E. coli?
Makes short primers that never leave the template
How many subunits does the core of E. coli polymerase have?
5 - 2 alpha-subunits, 1 beta, 1 beta’ and 1 omega
What is the sixth subunit of E. coli that is required for efficient initiation?
Sigma Factor
What is the complete enzyme called that starts E. coli RNA polymerase?
RNA polymerase holoenzyme
What are the three stages of RNA synthesis?
Initiation
Elongation
Termination
Where is RNA synthesis initiated?
At promoters
Where is the site of transcription?
5’ upstream of position of translational initiation codon in the gene
What is the transcription start site?
The first base copied on the template transcribed/sense strand
What is the number of the transcription start site?
+1
In reference to numbering, what are the values of the upstream bases?
Negative (-)
E. coli uses variations in __ and __ sequences to creat promoters of different efficiencies.
- 35
- 10
What does the commonality of promoter sequences suggest?
Functional importance
Where does the RNA polymerase holoenzyme first bind to the DNA in E. coli?
Weakly binds and slides to the -35 site; then binds tightly and forms a closed complex
After binding to the -35 site, RNA polymerase binds to the ___ site and unwinds how many base pairs?
-10 site
Unwinds about 17 base pairs; binds tightly to form open complex
What is the first nucleotide brought into the active site?
Almost always a purine
Why is a sigma subunit required?
To ensure specific, efficient recognition of the promoter
When is the sigma subunit lost?
Once several bases are incorporated
Why are there different sigma subunits?
To allow different promoters to be used
What facilitates the unwinding of DNA?
-ve supercoiling
At termination, why does RNA polymerase dissociate from the template?
It lacks a sigma subunit, not promoter and is not elongating a chain; it can’t bind to DNA without those things
What are the 2 mechanisms for transcriptional termination in prokaryotes?
Rho dependent
Rho independent
How does Rho dependent termination work?
Rho protein binds to poorly characterized sequences in the 3’ region of the transcript; uses ATP to break H bonds holding transcript to template
What drives Rho independent termination?
Secondary structure formation of RNA transcript and thermodynamics
In Rho independent termination, what reduces the base pairing template?
The region of transcript folds into a hairpin loop
After RNA forms a hairpin loop, what causes the DNA to rewind?
A U-rich environment that causes poor hydrogen bonding to template
In eukaryotes, typical mRNA promoter has what?
TATA box about 25 bases upstream of the +1 transcription start site
In eukaryotes, what does Pol II do?
Catalyzes RNA synthesis
In eukaryotes, what does TBP do?
Recognizes the TATA box
In eukaryotes, what does TFIIA do?
Stabilizes TFIIB and TBP binding to promoter
In eukaryotes, what does TFIIB do?
Binds TBP
Recruits Pol II-TFIIF complex
In eukaryotes, what does TFIIE do?
Recruits TFIIH
Has ATPase and helicase activities
In eukaryotes, what does TFIIF do?
Binds Pol II
Binds TFIIB and prevents nonspecific binding of Pol II
In eukaryotes, what does TFIIH do?
Unwinds DNA at promoter
Phosphorylates Pol II within CTD
What is a newly synthesized RNA molecule called?
Primary transcript
What are constitutive genes?
Genes that are involved in making things that we need all the time, like the enzymes of glycolysis and the TCA cycle
What are the core promotor proteins?
Pol II, TBP, TFIIA, TIFFB, TFIIE, TFIIF, TFIIH
What does TBP stand for?
TATA binding protein
What shape is tRNA usually in?
Cloverleaf
Where is the anticodon region of tRNA usually found?
Across from the “open”end
What is always found at the 3’ end of every tRNA?
A CCA
Why is it important to have a CCA on the 3’ end of tRNA?
That is where the amino acids bind
What does the tRNA anticodon base pair with?
The RNA template
RNA is transcribed as one big precursor. What happens to get all the forms of RNA?
Primary transcripts must be cleaved into functional fragments
Where are tandem arrays of rRNA found?
Nucleolar regions
When are tandem arrays of rRNA processed?
Post-transcriptionally
What chromosomes make up the nucleolar regions?
13-15, 21, 22
What is the “ribosomal factory”?
Nucleoli
After being arranged into subunits, where are rRNA and ribosomal proteins shipped?
To the cytoplasm
What 3 thing are required to make a mature mRNA?
5’ cap
3’ polyA tail
Introns spliced out
What is the poly A addition signal?
AAUAAA
When the PolyA addition signals is recognized by the enzyme complex, what happens?
The enzyme complex cleaves the transcript 10-30 nucleotides 3’ to signal
What tethers mRNA to CTD of Pol II?
Cap binding complex
In mRNA splicing, what is the cap tethered to?
RNA polymerase
What mediates mRNA splicing?
snRNA (called U1-U6)
snRNA’s come together to form what?
Spliceosome complex
How do mRNAs get spliced?
snRNAs build spliceosome, then bring in 5’ splice junction next to the branch point adenosine, form a loop, cut off both sides and allow them to ligate (I think)
What is usually larger, introns or exons?
Introns
Often, the introns spliced out are not junk. What are they/why are they spliced out?
Tissues can decide what do splice; usually introns that are spiced out are for use in different tissues
What type of RNA is most commonly edited?
In mitochondrial transcripts
What is rifamycin (rifampicin)?
Antibiotic that inhibits prokaryote RNA polymerase
How does rifamycin work?
Blocks the initiation of transcription
What does rifamycin treat?
TB
What does alpha-amanitin affect?
RNA Pol II
Blocks mRNA synthesis
