DNA/RNA Metabolism
Blackburn & Gait, Ch. 5
Transcription• understand components of transcription bubble• know the importance of promoter sequences in proks/euks• know how nucleic acid structure affects transcription termination (both Rho-dep and Rho-indep)• know an example of activator and repressor of transcription• know what transcriptional enhancer elements also called UAS (upstream activator sequence) do and their mode of action• understand attenuation
RNA Processing• understand 5’-capping reaction and reason for cap• understand 3’-endonuclease cleavage and polyadenylation and reason for A-tail• know steps in RNA splicing and alternative splicing
DNA metabolismReplicationDNA replication - process of copying genetic information DNA acts as a template for replication and transmission of genetic info
One strand is the complement of the other
DNA metabolismStages of ReplicationElongation
DNA metabolism
Stages of ReplicationElongation
RNA Metabolism
Transcription - uses DNA-dependent RNA polymerasePolymerization is “asymmetric” - only one strand of DNA used as template, new RNA chain is identical in sequence to the nontemplate strand
RNA Metabolism
TranscriptionRNA synthesis initiated at promoters (specific DNA sequence)Typical E.Coli promoters:
Pribnow box
-25 TATA box (TATAAA)
EUKARYOTES:
-75CAAT box
RNA MetabolismTranscription - terminationTermination sites have a palindromic sequence just prior to termination point and RNA transcribed has short inverted repeats (GC-rich regions) which form hairpinGC-rich regions followed by 4-10 A:T bpStem-loop structure in RNA being synthesized induces pausing of RNAPWeak U:A bp cause a conformational change in RNAP and RNA breaks free of DNARho-independent termination
RNA MetabolismTranscription - terminationRho-independent termination
RNA MetabolismTranscription - terminationRho-dependent terminationAlso get hairpin formed but not G-C rich and no uridines after it Special protein Rho is needed for terminationRho may bind 5’-end of nascent RNA and travel along behind RNAPRho may then interact with -subunit of RNAP and stop transcriptionRHO may be a helicase and unwind the RNA from the RNA-DNA hybrid
RNA MetabolismTranscription - terminationRho-dependent terminationAlso get hairpin formed but not G-C rich and no uridines after it Special protein Rho is needed for terminationRho may bind 5’-end of nascent RNA and travel along behind RNAPRho may then interact with -subunit of RNAP and stop transcriptionRHO may be a helicase and unwind the RNA from the RNA-DNA hybrid
RNA MetabolismTranscription - frequency of transcription regulated by inhibitors (repressors) and activators of RNAPRepressor = trp repressorActivator = CAP (catabolite activator protein)
DNA
Trp dimerHTH motif
Trp
DNA
CAP dimer (HTH motif)
cAMP
Interacts with RNAP
RNA MetabolismTranscriptionTranscriptional enhancer elements also called UAS (upstream activator sequence)If a UAS is deleted it abolishes promoter activity
RNA MetabolismTranscriptionTranscriptional enhancer elements also called UAS (upstream activator sequence)Enhancer function independent of orientation or distance from affected gene
RNA MetabolismTranscriptionTranscriptional enhancer elements also called UAS (upstream activator sequence)Models for action of enhancers
**Loop out DNA
Enhancer box is entry site, pro binds and then migrates along DNA in order to interact with RNAP
RNA MetabolismTranscriptionTranscriptional enhancer elements
RNA MetabolismTranscriptionTranscriptional enhancer elements
RNA MetabolismTranscriptionTranscriptional enhancer elements
Attenuation (prokaryotes)
• Attenuation is means of controlling transcription of a particular mRNA through the formation of translation-dependent alternative RNA structures
• For example, expression of the operon that encodes proteins required for tryptophan biosynthesis is modulated based on supply and demand for tryptophan
• A short open reading frame that encodes tryptophan, and a downstream RNA region (the attenuator) are key element of control
• Co-transcriptional translation determines what structure the attenuator RNA will adopt
• Abundant supply of tryptophan and movement of the ribosome promotes a structure that terminates transcription
• Stalling of ribosome due to lack of tryptophan promotes a structure that prevents termination
Attenuation (prokaryotes)
RNA processing
Proks vs. EuksIn proks, transcription & translation coupledIn euks, processes are temporally& spatially separated so more control
RNA processingmRNA processing:5’-capping3’-endonuclease cleavage and polyadenylationRNA splicing
RNA processingmRNA processing:5’-capping
RNA processingmRNA processing:3’-endonuclease cleavage & polyadenylation
RNA processingmRNA processing:RNA splicing & editing
RNA processingmRNA processing:RNA splicing & editing
RNA processing
Alternative splicing
RNA processing
Alternative splicing
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