1 / 60

Prokaryotic Transcription: Principles and Mechanisms

Explore the fundamentals of transcription in prokaryotes, including initiation, elongation, and termination processes. Learn about Escherichia coli RNA polymerase, promoters, and the synthesis of RNA from DNA templates. Delve into the role of RNA in protein translation and the factors influencing transcriptional and translational coupling. Understand essential components like RNA polymerase, rNTPs, and terminators. Gain insights into regulated steps in transcription processing and the unique features of prokaryotic transcription.

johntross
Download Presentation

Prokaryotic Transcription: Principles and Mechanisms

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. TRANSCRIPTION IN PROKARYOTES • Basic principles of transcription • Escherichia coli RNA polymerase • The E. colis70 promoter • Transcription in prokaryotes

  2. today’s focus DNA RNA PROTEIN

  3. Transcription of RNA (rRNA, tRNA, mRNA) • RNA transcribed from coding strand of DNA • RNA single stranded, may fold back on self and partially pair • RNA carries uracil, while DNA carries thymine • mRNA carries message to be translated into a polypeptide product • tRNA and rRNA function at the RNA level

  4. Potential Regulated Steps transcription processing cap AAAAAAAAAA export cap AAAAAAAAAA translation cap AAAAAAAAAA folding

  5. gene 1 gene 2 DNA transcriptional translational coupling (why wait?) • Many Pols simultaneously transcribing the gene • Ribosomes simultaneously translating protein • Does not occur in eukaryotes

  6. Transcription The synthesis of a single-stranded RNA from a double-stranded DNA template. RNA synthesis occurs in the 5’3’direction and its sequence corresponds to that of the DNA strand which is known as the sense strand. The template of RNA synthesis is the antisense strand. Necessary components: promoter/template, RNA polymerase, rNTPs, terminator/template

  7. 5’-CGCTATAGCGTTT-3’DNA nontemplate (+) strand 3’-GCGATATCGCAAA-5’DNA template (-) strand 5’-CGCUAUAGCGUUU-3’ RNA transcript (-) strand is antisense strand. (+) strand is sense strand

  8. Basic principles of transcription Initiation: polymerase and promoters Elongation: polymerase Termination: terminator

  9. Initiation:RNA polymerase is the enzyme responsible for transcription. It binds to specific DNA sequences called promoters to initiate RNA synthesis. These sequences are upstream (to the 5’ end) of the region that codes for protein, and they contain short, conserved DNA sequences which are common to different promoters. The RNA polymerase binds to the dsDNA at a promoter sequence, resulting in local DNA unwinding. The position of the first synthesized base of the RNA is called the start site and is designate as position +1

  10. +1 Promoter Initiation • Binding of an RNA polymerase to the dsDNA • Slide to find the promoter • Unwind the DNA helix • Synthesis of the RNA strand at the Start site (initiation site), this position called position +1

  11. Elongation: RNA polymerase moves along the DNA and sequentially synthesizes the RNA chain. DNA is unwound ahead of the moving polymerase, and the helix is reformed behind it.

  12. Elongation • Add ribonucleotides to the 3’-end • The RNA polymerase extend the growing RNA chain in the direction of 5’ 3’ • The enzyme itself moves in 3’ to 5’ along the antisense DNA strand.

  13. Termination: RNA polymerase recognizes the terminator which causes no further ribonucleotides to be incorporated. This sequence is commonly a hairpin structure. Some terminators require an accessory factor called rho for terminaton • The dissociation of the transcription complex from the template strand • Occurring at the terminator

  14. Termination site for RNA polymerase in bacteria • Direct termination: The RNA hairpin loop of GC sequences and section of U residues appear to serve as signal for RNA polymerase release and termination of transcription. • Alternative termination method: rho protein binds to specific sequences referred to as rut.rho pulls RNA polymerase off RNA.

  15. DNA helix is transiently unwound within the polymerase while mRNA is synthesized polymerase moves downstream As it synthesizes mRNA Note: unlike helicase, ATP not required

  16. Transcription

  17. RNA polymerase: synthesis of RNA strand from DNA template. 1.Requires no primer for polymerization 2.Requires DNA for activity and is most active with a double-stranded DNA as template. 5’  3’ synthesis (NMP)n + NTP  (NMP)n+1 + PPi 3. Require Mg2+ for RNA synthesis activity

  18. 4. All RNA polymerases lack 3’  5’ exonuclease activity, and one error usually occurs when 104 to 105 nucleotides are incorporated. The polymerases of bacteriophage T3 and T7 are smaller single polypeptide chains, they synthesize RNA rapidly (200 nt/sec) and recognize their own promoters which are different from E. coli promoters. 5. usually are multisubunit enzyme, but not always.

  19. 6. Different from organism to organism 7. E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA.

  20. E. coli RNA polymerase E.Coli RNA polymerase RNA polymerase is responsible for RNA synthesis (Transcription). The core enzyme, consisting of 2, 1 , 1 ’,and 1  subunits, is responsible for transcription elongation. The sigma factor (), is also required for correct transcription initiation. The complete enzyme, consisting of the core enzymes plus the factor , is called the holoenzyme.

  21. E. coli RNA polymerase 155 KD 36.5 KD 11 KD 36.5 KD 70 KD Initiation only 151 KD Both initiation & elongation

  22. Shaped as a cylindrical channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp. • RNA synthesis rate: 40 nt per second at 37oC

  23. RNA polymerase

  24. E. coli polymerase: a subunit • Two identical subunits in the core enzyme • Required for core protein assembly ,but has no clear function • May play a role in promoter recognition. Once the subunit modified, it reduces the binding affinity to promoter region.

  25. E. coli polymerase: b subunit • The catalytic center of the RNA polymerase • Rifampicin：has been shown to bind to the β subunit, and inhibit transcription initiation by prokaryotic RNA pol. Mutation in b gene can result in rifampicin resistance.

  26. E. coli polymerase: s factor Sigma factor is a separate component from the core enzyme. E. coli encodes several s factors, the most common being s70. A s factor is required for initiation at the correct promoter site. It does this by decreasing binding of the core enzyme to nonspecific DNA sequences and increasing specific promoter binding. The s factor is released from the core enzyme when the transcript reaches 8-9 nt in length.

  27. E. coli polymerase: s factor • Many prokaryotes contain multiple s factors to recognize different promoters. The most common s factor in E. coli is s70. • Binding of the s factor converts the core RNA pol into the holoenzyme. • s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites (104) and increasing the affinity for the corresponding promoter • s factor is released from the RNA pol after initiation (RNA chain is 8-9 nt) • Less amount of s factor is required in cells than that of the other subunits of the RNA pol.(30% present)

  28. The E coli σ70 promoter • Promoter sequences: • -10 sequence and -35 sequence • 2. Transcription Start site • 3. Promoter efficiency

  29. Promoters: contain conserved sequences which are required for specific binding of RNA pol and transcription initiation +1 promoter Transcribed region terminator ATACG TATGC DNA Antisense strand Transcription RNA Roles in transcription: different promoters result in differing efficiencies of transcription initiation, which in turn regulate transcription.

  30. Promoter sequence • Lies upstream of the start site of transcription ( position +1),thus the promoter sequences are assigned a negative number • Contains short conserved sequences critical for specific binding of RNA polymerase and transcription initiation

  31. s70 promoter ---5-8 bp--- G C T A TTGACA -----16-18 bp------- TATAAT -35 sequence -10 sequence +1 • Consists of a sequence of between 40 and 60 bp • -55 to +20: bound by the polymerase • -20 to +20: tightly associated with the polymerase and protected from nuclease digestion by DNaseΙ • up to position –40: critical for promoter function • -10 and –35 sequence:particularly important for promoter function

  32. -10 sequence (Pribonow box) • 6 bp sequence which is centered at around the –10 position • A consensus sequence of TATAAT • The first two bases(TA) and the final T are most highly conserved among other E. coli promoters • This hexamer is separated by between 5 and 8 bp from position +1,and the distance is critical

  33. -35 sequence: enhances recognition and interaction with the polymerase s factor • A conserved hexamer sequence around position –35 • A consensus sequence of TTGACA • The first three positions (TTG) are the most conserved among E. coli promoters. • Separated by 16-18 bp from the –10 box in 90% of all promoters

  34. Transcription start site The sequence around the start site influences initiation • Is a purine in 90% of all genes • G is more common at position +1 than A • Often, there are C and T bases on either side of the start site nucleotide(i.e.CGT or CAT)

  35. The sequences of five E. coli promoters

  36. E. coli promoter sequences for 13 genes • Promoter sequences for mRNAs. RNA polymerase seeks out the consensus sequences for proper orientation for binding to initiate transcription. • Note promoter sites have regions of similar sequences at the -35 region and -10 region. • Minus numbers represent bases upstream of mRNA start point, +1 is the first base in the RNA transcript.

  37. Promoter structure in prokaryotes T T G A C A T A T A A T 82 84 79 64 53 45% 79 95 44 59 51 96% mRNA PuPuPuPuPuPuPuPu AUG 5’ -30 -10 +1 [ ] Promoter transcription start site mRNA 5’ -35 region -10 region TTGACA TATAAT AACTGT ATATTA -36 -31 -12 -7 +1 +20 Pribnow box consensus sequences

  38. trancription initiation, elongation and termination Promoter binding DNA unwinding RNA chain initiation RNA chain termination RNA chain elongation Rho-dependent termination

  39. Promoter binding The σ factor enhances the specificity of the core polymerase for promoter binding. The polymerase finds the promoter –35 and –10 sequences by sliding along the DNA and forming a closed complex with the promoter DNA

  40. Binding

  41. Unwinding Initiation Elongation termination

  42. E. coli RNA polymerase +  subunit  (sigma subunit) allows RNA polymerase to recognize and bind specifically to promoter regions.

  43. Promoter binding • The σ factor enhances the specificity of the core α2ββ’ωRNA polymerase for promoter binding • The polymerase finds the promoter –35 and –10 sequences by sliding along the DNA extremely rapidly and forming a closed complex with the promoter DNA (The initial complex of the polymerase with the base-paired promoter DNA)

  44. DNA unwinding : around 17 bp of the DNA is unwound by the polymerase, forming an open complex. DNA unwinding at many promoters is enhanced by negative DNA supercoiling. However, the promoters of the genes for DNA gyrase subunits are repressed by negative supercoiling.

  45. DNA unwinding • It is necessary to unwind the DNA so that the antisense strand to become accessible for base pairing, which is carried out by the polymerase. • Negative supercoiling enhances the transcription of many genes but not all by facilitating unwinding . • The initial unwinding of the DNA results in formation of an open complex with the polymerase and this process is referred to as tight binding

  46. RNA chain initiation • No primer is needed • Start with a GTP or ATP • The first 9 nt are incorporated without polymerase movement along the DNA or σfactor release • The RNA polymerase goes through multiple abortive chain initiations, which are important for the overall rate of transcription • The minimum time for promoter clearance is 1-2 seconds (a long event)

  47. RNA chain elongation • σFactor is released to form a ternary complex of the newly synthesized RNA, causing the polymerase to progress along the DNA (promoter clearance) • Transcription bubble (unwound DNA region, ~ 17 bp) moves along the DNA with RNA polymerase which unwinds DNA at the front and rewinds it at the rear. 10-12 nucleotides at the 5’-end of the RNA are constantly base-paired with the DNA template strand. • The E. coli polymerase moves at an average rate of ~ 40 nt per sec, depending on the local DNA sequence.

More Related