RNA is synthesized by ___ ___. It requires a ____ template and synthesizes new RNA 5’ to 3’. No ____ is needed to initiate RNA synthesis. RNA polymerases do no _____
RNA polymerase; DNA; primer; proofread
E. coli uses a single RNA polymerase for all transcript synthesis:
rRNA, tRNA, and mRNA
Core enzyme of E. coli RNA polymerase is inefficient at initiating RNA synthesis and consists of 5 subunits. The sixth subunit ___ is req for efficient initiation and the complete enzyme is then called the RNA polymerase ____
- 2 alpha subunits
- 1 beta
- 1 beta prime
- w (gamma)
sigma factor; holoenzyme
Transcription is initiated at ____ sites upstream. Things that are upstream have ____ numbers. The first base on the template transcribed strand copied is called the _____ ___ ___.
promoter; negative; transcription start site
What are the E. coli promoter consensus sequences
regions within promoters that have similar DNA sequences…centered around -35 and -10 region (pribnow box)
what does commonality of seq suggest?
functional importance for the regions and binding RNA polymerase for that region.
RNA polymerase binds weakly to DNA until it binds to -35 site and closes tightly. RNA polymerase then moves forward to -10 site and unwinds about ____ bp in front and forms open complex.
What is the first nucleotide brought into active site
a purine; pyrimidine on template
Oncle nucleotides are brought in ____ units fall off.
As elongation of transcript goes forward; DNA helix gets unwound in front of enzyme and rewinds behind which displaces growing RNA strand. At any one point, about ____ bp unwound
RNA polymerase dissociates from the template. Where does it terminate?
3’ downstream of translation termination codon.
what are the two mechanisms for transcriptional termination in E. coli?
- Rho dependent
2. Rho independent
How does Rho dependent termination occur
- Req ATP
- Binds to seq in 3’ untranslated region of mRNA and uses energy from ATP to break H bonds holding transcript to template.
how does Rho independent termination occur
driven by secondary structure formation of RNA transcript and thermodynamics. Region of transcript folds into hairpin loop and reduces bp to template. This region following the loop is very U rich which favors DNA rewinding bc there is poor H bonding
Eukaryotes have ____ diff kinds of RNA polymerases
I: in nucleolus; transcribes rRNA
II: in nucleus; transcribes mRNA. extremely sensitive to alpha amanitin
III: in nucleus; transcribes tRNA & 5S rRNA
In eukaryotes, typical mRNA promoter has ____box about ____ bases upstream of transc start site which is the core promoter.
RNA pol II catalyzes RNA synthesis. ____ recognizes TATA box. ____ stabilizes TFIIB and TBP binding to promoter. TFIIB binds ___ and recruits more Pol II-TFIIF complex. _____ recruits TFIIH. _____ binds Pol II and binds TFIIB and prevents nonspecific binding of Pol II. _____ unwinds DNA at promoter; phosphorylates Poll II within CTD (carboxy terminal domain)
TBP; TFIIA; TBP; TFIIE; TFIIF; TFIIH
How is RNA processed? what is a newly synthesized RNA molecule called
Prokaryote mRNAs are ready to be used in translation w/o furhter modification. primary transcript.
tRNA and rRNA never become _____. tRNA’s undergo ____ modifications and rRNAs are involved in cleavage of large primary trancript into functional fragments.
tRNA is single strand of RNA that base pairs with itself and makes unusual structures. Always a piece at 5’ end that gets removed by RNAse P. Always a piece at 3’ end that gets removed and replaced with a ____and where aa bind.
rRNA is transcribed as a big precursor molecule. RNA cleavage occurs to give each RNA _____
intermediate (ind compennts of rRNA and tRNA)
Where are tandem arrays of rRNA genes found?
found in nucleolar regions of 13, 14, 15, 21, and 22.
In eukaryotes _____ also gets processed, but NOT in prokaryotes
mRNA; 5’ cap to 5’ end and 3’ polyA tail and splicing of introns
hetero nuclear RNA means
it hasnt been processed yet
processing of mRNA happens concurrently with:
what is added to 5’ cap
7 methylguanosine adds to 5’ end to the transcript in opp orientation. Helps mRNA transcript stay in there longer.
Cap binding complex
recognizes new cap and keeps it tethered. So as its transcribed intron can get spliced out and poly A tail can be added on the end.
What is splicing mediated by? What is its role?
small nuclear RNAs (snRNAs) which form splicesome complex. They help bring 5’ splice junction and branch point close to each other. Form the loop. Cuts off one side and then the other side and helps ligate the exons.
Introns always have GU at 5’ end and AG at 3’ end. What does the branch point help do?
Branch point helps make lariat structure.
What do the OH groups serve as
nucleophiles to first form the lariat structure, then join the donor and acceptor junction.
____ and transcription are coordinated
____ are larger than the exons
____ splicing is often tissue dependent
Some mRNAs are edited before translation. Alteration usually involves enzymatic deamination of A or C to form inosine or ____. In intestine, deamination converts ____ to uridine.
what are differences in RNA processing in eukaryotes vs prokaryotes
- No 5’ cap 3’ poly a tail or splicing of mRNA in prokaryotes.
- There is no removal of UU at 3’ end for tRNA in prokaryotes.
- There is some methylation of some bases in rRNA in prokaryotes whereas in eukaryotes, theres methylation of some riboses
what are inhibitors of transcription
- Rifamycin: antibiotic that inhibits prokaryote but not eukaryote RNA polymerase.
- alpha amanitin: toxin from poisonous mushroom that mainy affects RNA pol II, blocks mRNA synthesis
- Actinomycin D: planar molecule intercalates into double helix bet two GC base pairs which blocks polymerase movement.
- acridine: research reagant that works in similar manner.