L2: Coupling and small regulatory RNA's in Prokaryotes Flashcards
What allows the coupling of transcription and translation in Prokaryotes but not Eukaryotes?
- In Prokaryotes, the two machineries both exist in the cytoplasm whereas in Eukaryotes they are spatially separated.
- This allows the expressome to assemble
Define ‘Expressome’. What is its function?
- The expressome is the name for the bound RNA Polymerase and Ribosome in coupling
- It allows the tight binding of transcription and translation
Define ‘cistron’
Alternative word for gene, emphasises particular polypeptide
How does the intrinsic model of termination operate?
An intrinsic termination signal is present in the transcript, consisting of a GC-rich hairpin followed by a run of U residues
How is polycistronic transcription coded for in prokaryotes?
- Each ORF is preceded by an RBS (aka SD) where the ribosome can associate -> enables simultaneous translation of multiple proteins from one mRNA
How is the ribosome produced?
- Transcribed as one transcript -> 5S and 23S, as well as 16S
- Cleaved by specific RNAse III
- Components translated -> 50S subunit and 30S -> assembled ribosome (70S)
- S = Svedburg unit, sedimentation coefficient
How does the ribosome recognise DNA?
- RBS/SD site is an AG-rich region 6-8 nts upstream of initiating codon
- This sequence is complementary to the 3’ end of the 16S RNA
Attenuators: What are they? Key example in prokaryotes:
- Regulate termination of transcription
- Usually located prior to the genes they control; act to prevent transcription by premature termination in given circumstances
- e.g TrpL leader sequence contains attenuator region
How does the attenuator work in Trp operon?
- TrpL sequence is rich in certain AAs; preceded by attentuator sequence which froms two mutually exclusive stem loop secondary structures
- High [Trp] -> 3:4 loop, attenuated transcript
- Low [Trp] -> Stalled at site 1, 2:3 loop permitted -> continues transcribing to produce Trp
3 key ways that translation can be regulated (with examples):
- Translation of mRNA blocked by regulator protein binding mRNA sequence, occluding either AUG, SD or both e.g. T4 p32 (phage repressor protein 32)
- Changes in secondary structure in mRNA itself permit or prevent translation e.g. RNA phage cistrons, were each is expressed in order due to ‘untangling’ as the ribosome proceeds
- Translation of r-protein operons controlled by a product of the operon binding to a site on polycistronic mRNA e.g. spc operon (S8 able gene able to autogenously regulate entire operon)
How may transcription/translation be targeted pharmaceutically?
Give 3 examples with mechanisms
- Several antibiotics target these processes
- e.g. Rifampin (TB treatment) blocks RNA synthesis
- Erthyromycin blocks peptide elongation by binding 50S subunit
- Tetracycline blocks tRNA by binding 30S subunit
How is mRNA ‘cleaned up’ after transcription?
- mRNA is degraded rapidly in a matter of minutes by ribonuclease
- Occurs from 5’ end
2 basic mechanisms of sRNA action:
- Protein binding -> modulation of protein activity
- Antisense sRNAs -> translation activation or inhibition, and/or mRNA degradation
3 modes for sRNA regulating transcription:
- sRNA forms duplex in protein binding site on mRNA -> protein unable to bind
- sRNA binds specific site on mRNA to target it for attack by endonucleases
- rRNA binding to mRNA prevents secondary structure formation -> indirect effect on function
Example: Transcriptional regulation by prompting endonuclease attack (by sRNAs)
- Reaction of ‘Fur’ to low and high [Iron]
- High [Iron] -> Fur binds and represses transcription of ent and rhyB, mRNA of succinate dehydrogenase unbothered
- Low [Iron] -> Fur dissociates, ent and rhyB transcribed -> sRNA binds succinate dehydrogenase transcript and targets for RNase
