Chapter 26 RNA Metabolism 1. How is rna synthesized using dna templates (transcription )? 2. How is newly synthesized primary rna transcripts further processed to make functionalrna molecules? 3. How is rna and dna synthesized using rna as template(reverse transcription) 4. What is the evolutionary implication of the structural and functional complexity of rna molecules
Chapter 26 RNA Metabolism 1. How is RNA synthesized using DNA templates (transcription)? 2. How is newly synthesized primary RNA transcripts further processed to make functional RNA molecules? 3. How is RNA and DNA synthesized using RNA as template (reverse transcription); 4.What is the evolutionary implication of the structural and functional complexity of RNA molecules?
1 RNA molecules have great structural and functional diversity With structures comparable to proteins in complexity and uniqueness o Function as messengers between DNa and polypeptides(mRNA), adapters(tRNA) to match a specific amino acid with its specific genetic code carried on mRNA, and the structural and catalytic components of the protein-synthesizing ribosomes (rRNA o Stores genetic information in RNa viruses o Catalyzes the processing of primary rna transcripts o Might have appeared before dna during evolution
1. RNA molecules have great structural and functional diversity ⚫ With structures comparable to proteins in complexity and uniqueness. ⚫ Function as messengers between DNA and polypeptides (mRNA), adapters (tRNA) to match a specific amino acid with its specific genetic code carried on mRNA, and the structural and catalytic components of the protein-synthesizing ribosomes (rRNA). ⚫ Stores genetic information in RNA viruses. ⚫ Catalyzes the processing of primary RNA transcripts. ⚫ Might have appeared before DNA during evolution
2. DNA and RNA syntheses are similar in some aspects but different in others e Similar in fundamental chemical mechanism both are guided by a template, both have the same polarity in strand extension(5 to 3); both use triphosphate nucleotides (dnTP or nTP) o Different aspects: No primers are needed; only involves a short segment of a large DNA molecule; uses only one of the two complementary dna strands as the template strand no proofreading, subject to great variation(when, where and how efficient to start)
2. DNA and RNA syntheses are similar in some aspects but different in others ⚫ Similar in fundamental chemical mechanism: both are guided by a template; both have the same polarity in strand extension (5` to 3`); both use triphosphate nucleotides (dNTP or NTP). ⚫ Different aspects: No primers are needed; only involves a short segment of a large DNA molecule; uses only one of the two complementary DNA strands as the template strand; no proofreading; subject to great variation (when, where and how efficient to start)
3. The multimeric RNA polymerase in E, coli has multiple functions The holoenzyme consists of five types of subunits (a2BBo)and its is used to synthesize all the rna molecules in e coli The multiple functions include searches for initiation sites on the dna molecule and unwinds a short stretch of DNa(initiation) selects the correct NTP and catalyzes the formation of phosphodiester bonds(elongation) detects termination signals for RNa synthesis (termination)
3. The multimeric RNA polymerase in E.coli has multiple functions ⚫ The holoenzyme consists of five types of subunits (a2bb’ s)and its is used to synthesize all the RNA molecules in E. coli. ⚫ The multiple functions include: – searches for initiation sites on the DNA molecule and unwinds a short stretch of DNA (initiation); – selects the correct NTP and catalyzes the formation of phosphodiester bonds (elongation); – detects termination signals for RNA synthesis (termination)
Enzyme assemblys promoterrecognition, PoSsible catalytic subunits activator binding a BB′o Role unknown (not needed in vitro 5115511kDa a 35.5kDa Promoter U(3290kDa) specificit The e coli rna polymerase holoenzyme consists of six subunits:a2ββB0σ
The E. coli RNA polymerase holoenzyme consists of six subunits: a2bb’ s. Possible catalytic subunits Promoter specificity Enzyme assembly, promoter recognition, activator binding Role unknown (not needed in vitro) 36.5 kDa 151 155 11 kDa (32-90 kDa)
4. RNA synthesis occurs in a moving transcription bubble on the DNA template o Only a short RNA-Dna hybrid(8 bp in bacteria) is present through the transcription process At each moment, a region of about l7 bp on the e coli dna is unwound in the transcription bubble The rna chain is extended at a rate of 50-90 nucleotides/ second by the E coli rna polymerase o Unwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the dna (relieved by the action of topoisomerases)
4. RNA synthesis occurs in a “moving” transcription bubble on the DNA template ⚫ Only a short RNA-DNA hybrid (~8 bp in bacteria) is present through the transcription process. ⚫ At each moment, a region of about 17 bp on the E. coli DNA is unwound in the transcription bubble. ⚫ The RNA chain is extended at a rate of 50-90 nucleotides/second by the E. coli RNA polymerase. ⚫ Unwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the DNA (relieved by the action of topoisomerases)
Transcription bubble Nontemplate strand RNA polymerase Rewinding Unwinding DNA 3 3 stran d RNA 6′RNA-DNA Active site hybrid, 8 bp Direction of transcription (5)CGCTATAGCGTTT(3) DNA nontemplate(coding)strand (3)GCGATATCGCAAA(5) DNA template strand (5) CGCUAUAGCGUUU(3) RNA transcript
Negative supercoils Positive supercoilS 5 RNA Direction of transcription (b)
5. RNA polymerase recognizes specific promoter sequences on DNA to initiate transcription o Promoter sequences are located adjacent to genes o Promoters can be identified using" protection assays (e.g, footprinting techniques) o Promoters, although all bind to the same polymerase have quite variable DNa sequences(surprisingly), but with two consensus sequences centered at-10 and 35 positions (the first residue of the rna is given +1) o Promoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations and activity comparison)
5. RNA polymerase recognizes specific promoter sequences on DNA to initiate transcription ⚫ Promoter sequences are located adjacent to genes. ⚫ Promoters can be identified using “protection assays” (e.g., footprinting techniques). ⚫ Promoters, although all bind to the same polymerase, have quite variable DNA sequences (surprisingly), but with two consensus sequences centered at –10 and – 35 positions (the first residue of the RNA is given +1). ⚫ Promoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations and activity comparison)
The footprinting P end-labeled DNA technique Specific binding protein protein DNase nicks DNase nicks randoml !!!!!! ↓⌒↓↓↓ The footprint 32P-labeled Gel electrophoresis 32P-labeled fragments pattern fragments
The footprinting technique The footprint - protein + protein randomly
