3. DNATranscription in Prokaryotes
•First stage of gene expression.
•Occurs in cytoplasm.
•By DNA - dependent RNA Polymerase.
•Synthesis of RNA from DNA.
• rRNA, mRNA, tRNA, etc
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4. RNA Pol
• Coenzyme.
• Holo if coupled with sigma
factor (transcription
initiation factor).
• Core cmplx has five
subunits.
• Subunits assemble prior to
transcription.
• Disassemble after
transcription.
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5. RNA Pol
• Two α: to assemble RNA
Pol on DNA.
• β: adheres with
ribonucleoside triphosphate
of nascent mRNA.
• βʹ: attaches to the template
DNA strand.
• ω: facilitates RNA Pol
assembly.
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7. Prokaryotic Promoter Region
• Promoter: region on DNA with particular sequence where RNA Pol binds
to.
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Mostly exists upstream of
gene to be transcribed.
• -35 and -10 regions are
similar across promoters.
• They form consensus
sequences
Rate Initiation 5' 3'
8. 1. Initiation
•Sigma factor binds to promoter region.
•Core enzyme causes DNA unwinding at transcription
start site, facilitated by the -10 region (Pribnow box).
•Abortive initiation: RNA Pol aborts when it fails to
synthesize beyond threshold level (10 nucleotides).
•Successful initiation: RNA Pol stabilizes and goes beyond
threshold to initiate transcription.
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10. 2. Elongation
• Begins at +1 site.
• Release of σ subunit from the RNA Pol allows core E to
proceed along the DNA.
• RNA Pol opens DNA, stabilizes ssDNA and unwinds DNA.
• Reads the template DNA strand in 3ʹ to 5ʹ.
• Synthesizes RNA from 5ʹ to 3ʹ.
• Approx 40 nucleotides per sec.
• RNA can form all the RNAs needed (rRNA, mRNA or tRNA).
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14. 3. Termination in prokaryotes
•Either of the following:
1. ρ (Rho) protein independent
•Physical RNA Pol modification terminates
transcription.
2. ρ (Rho) protein dependent
•Rho protein and helicase protein terminates
transcription.
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15. a. Rho independent termination
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Also called intrinsic termination.
Termination site is G-C rich.
18. Rho independent termination
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mRNA dissociates
Terminating transcription
DNA strands zip up behind RNA Pol III
Due to more stable A-T bonds
Between template and non-template strands.
19. b. Rho dependent termination
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They never meet until …
20. Rho dependent termination
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RNA Pol reaches termination site and stalls,
Then Rho catches up with RNA Pol.
Using ATP-dependent helicase activity, Rho unwinds
mRNA from template strand.
Rho reaches termination site and
Facilitate dissociation of
Transcription machinery
21. Initiation in Eukaryotes
•Requires RNA Pol and transcription factor (TF).
•Three different RNA Pols required.
i. RNA Pol I for rRNA
ii. RNA Pol II for mRNA
iii. RNA Pol III for tRNA
• One commonTF is TF2D.
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22. Initiation in Eukaryotes
•TF binds to promoter region for RNA Pol to bind to and
read the DNA.
•Promoter regions include:
i. TATA box (at -25 from starting +1 site)
ii. CAAT box
iii. GC box
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Remember
-35
-10
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ThenTFIIB binds toTFIID (or
to RNA Pol II to act as a
bridging protein).
TFIIA binds and stabilizes
TFIID-TATA box interaction.
Initiation in Eukaryotes
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Initiation in Eukaryotes
Thus RNA Pol II (already complexed to
TFIIF) now binds.
This follows binding ofTFIIE andTFIIH.
This forms transcription initiation complex.
TFIIB recruits Pol II-TFIIF complex
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Transcription termination in Eukaryotes
At the end of a gene, RNA Pol II encounters poly-A
signal sequenceTTATTT.
Polyadenylation – lots of adenines.
Cleavage & polyadenylation specificity factor
Cleavage stimulatory factor
30. Transcription termination in Eukaryotes
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It triggers transfer of CPSF and CstF to the RNA.
Then additional cleavage factors are recruited.
32. RNA Processing
• The mRNA produced needs to be modified to be matured.
• Otherwise it is heterogeneous nuclear RNA (hnRNA) that needs processing.
• Post-transcription processing converts primary transcript into functional
RNAs.
• Briefly, lets dive into that.
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33. RNA Processing
•Modified through:
1. Cleavage
•Larger RNA precursors are cleaved to form smaller
RNAs.
•Primary transcript is cleaved by ribonuclease – P to
form 5 – 7 tRNA precursors.
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34. RNA Processing
2. Capping and tailing
• At 5ʹ end, a 7-methyl guanosine (7mG), a modified GTP cap, is
added.
• Capping initiates translation.
• It also prevents degradation by nuclease enzymes.
• A poly-A tail is added at the 3ʹ end, sometimes with 2000 adenine
nucleotides.
• Tailing is for same fns as capping.
• It also helps in transporting mRNA out of nucleus.
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35. RNA Processing
3. Splicing
•Primary RNA has two segments; non-coding introns and
coding exons.
•Introns are spliced out using ATP.
•Adj exons are joined to produce mature mRNA.
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37. RNA Processing
4. Nucleotide modifications
•Most common in tRNA – methylation.
•Eg methyl cytosine, methyl guanine).
•Deamination eg inosine from adenine.
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38. Prokaryotic vs EukaryoticTranscription
Prokaryotes Eukaryotes
Transcription Occurs in cytoplasm Occurs in nucleus
Transcription & Translation Occur simultaneously Don’t occur simultaneously
RNAs Released & processed in cytoplasm In nucleus
Transcriptional factors Not needed (sigma) Needed
RNA Pol One only Three (I, II and III)
RNA Pol Complex of five polypeptides 10 – 15
Promoter region Always upstream of start site May be downstream eg for RNA III
Termination involving rho yes No
Post transcriptional modification no Yes
Introns in mRNA absent present
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39. Prokaryotic vs EukaryoticTranscription
•DNA acts as a template for RNA synthesis.
•Chemical composition of the transcript is similar.
•RNA Pol facilitates transcription in both.
•Three steps in both.
•Etc
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