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