原核生物转录

RNA polymerase differs from organism to organism
Section K: Transcription in prokaryotes
K1 Basic principles of transcription
An overview, the process of RNA synthesis ( initiation, elongation, termination)
Link
Elongation
• Covalently adds ribonucleotides to the 3’-end of the growing RNA chain. • 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.
Primary transcript is the original unmodified RNA product corresponding to a transcription unit.
Replication: synthesis of two DNA molecules using both parental DNA strands as templates. Duplication of a DNA molecule.
RNA Terminator is a sequence of DNA, represented at the end of the transcript, that causes RNA polymerase to terminate transcription.
RNA polymerases are enzymes that synthesize RNA using a DNA template (formally described as DNA-dependent RNA polymerases).
E. coli RNA polymerase
155 KD 36.5 KD 11 KD 36.5 KD 151 KD 70 KD
Initiation only
Both initiation & elongation
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.
Replication-synthesis of the leading strand
the same direction as the replication fork moves
Replication- Synthesis of the Okazaki fragments
Opposite to the replication fork movement
Terminator structure
K2 Escherichia coli RNA polymerase
1. E. coli RNA polymerase 2. a subunit 3. b subunit 4. b’ subunit 5.sigma (s) factor
K2-1 E. coli RNA polymerase
Transcription
Fig. 2. Structure of a typical transcription unit
Initiation (template recognition)
1. Binding of an RNA polymerase to the dsDNA 2. Slide to find the promoter 3. Unwind the DNA helix 4. Synthesis of the RNA strand at the start site (initiation site), this position called position +1
Coupling the synthesis of leading and lagging strands with a dimeric DNA pol III (E. coli)
Transcription
1. RNA synthesis occurs in the 5’3’ direction and its sequence corresponds to the sense strand (coding strand). 2. The template of RNA synthesis is the antisense strand (template strand). 3. Phosphodiester bonds: same as in DNA 4. Necessary components: RNA polymerase, transcription factors, rNTPs, promoter & terminator/template
Link
Termination
• Ending of RNA synthesis: the dissociation of the RNA polymerase and RNA chain from the template DNA at the terminator site. • Terminator: often contains selfcomplementary regions which can form a stem-loop or hairpin structure in the RNA products (see K4 for details)
K2 Escherichia coli RNA polymerase
Properties, a subunit, b subunit, b’ subunit, sigma (s) factor
K3 The E. coli s70 promoter
Promoter, s70 size, -10 sequence, -35 sequence, transcription start site, promoter efficiency
Synthesis of single-stranded RNA from DNA template.
RNA polymerase
1. Requires no primer for polymerization 2. Requires DNA for activity and is most active with a double-stranded DNA as template. 3. 5’ 3’ synthesis 4. Require Mg2+ for RNA synthesis activity 5. lacks 3’ 5’ exonuclease activity, and the error rate of nucleotides incorporation is 10-4 to 10-5. 6. usually are multisubunit enzyme.
Upstream identifies sequences proceeding in the opposite direction from expression; for example, the bacterial promoter is upstream from the transcription unit, the initiation codon is upstream of the coding region. Transcription unit is the distance between sites of initiation and termination by RNA polymerase; may include more than one gene. Promoter is a region of DNA involved in binding of RNA polymerase to initiate transcription
E. coli polymerase
1. E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. 2. One of the largest enzyme in the cells 3. Consists of at least 5 subunits in the holoenzyme, 2 alpha (a), and 1 of beta (b), beta prime (b’), omega (w) and sigma (s) subunits 4. Shaped as a cylindrical （圆柱形）channel that can bind directly to 16 bp of DNA. The whole polymerase binds over 60 bp. 5. RNA synthesis rate: 40 nt per second at 37oC
K1-1: Transcription: an overview
Key terms defined in this section (Gene VII)
+1
upstream
m7Gppp
Gene X
downstream
Primary transcript
AAAAAn
mRNA
Coding strand of DNA has the same sequence as mRNA. Downstream identifies sequences proceeding further in the direction of expression; for example, the coding region is downstream of the initБайду номын сангаасation codon.
K4 transcription process.
Promoter binding, unwinding, RNA chain initiation, elongation, termination (r factor)
K1: Basic principles of transcription
1. Transcription: an overview (comparison with replication) 2. The process of RNA synthesis: initiation, elongation, termination
1 DNA molecule 2 DNA molecules
Transcription: synthesis of one RNA molecule using one of the two DNA strands as a template.
1 DNA molecule 1 RNA molecule
K1-2: The process of RNA
synthesis 1.initiation
2.elongation 3.termination
Flowchart of RNA synthesis
Back 1, 2
+1 Promoter
DNA RNA
Terminator Sense strand Transcribed region Antisense strand