L13 : Coupling of Transcription and Translation Flashcards
What are prokaryotic strategies?
Majority of all life on Earth
Unicellular
Adaptive, fast responses to global stimuli
Growth/cell division tightly linked to nutrient availability
Chronic energy stress needs tightly regulated resource economy
What are complex eukaryotic strategies?
Smaller biomass fraction
Multicellular
Specialisation, slow responses requiring complex communication and nutrient supply
Majority of cells do not divide frequently
What is the stringent response
Response to nutrient starvation eg. amino acids (common for microbes in natural environments)
Downregulation of translation machinery and ribosomal protein genes (protein synthesis + growth stops)
Upregulation of amino acid biosynthesis, proteolysis, stress factor genes (cells compensate for starvation)
Functional method to prevent production of mRNA that cannot be translated
How is starvation detected in stringent response?
During AA starvation, aminoacyl tRNA synthetases cannot charge tRNAs with AAs
Empty tRNAs loaded onto ribosome causing stalling (idling reaction/hungry codons) and recruitment of RelA
What is the pathway of RelA within stringent response?
RelA associates to stalled ribosome via L11
Triggers synthesis of pppGpp from ATP and GTP, which quickly hydrolyses itself into ppGpp
SpoT protein then able to degrade ppGpp to GDP + PPi
What is the role of ppGpp in stringent response?
ppGpp is alarmone - small second messanger molecule from ribosomes to RNAP
ppGpp binding to RNAP changes promoter usage with stringent (containing a discriminator region) promoters downregulated whilst relaxed promoters upregulated
Altered gene expression induces AA biosynthesis
How is RNAP negatively controlled by ppGpp?
ppGpp destabilises open complex due to discriminator within stringent promoter
Relaxed promoter resistant to ppGpp so unaffected
How is RNAP positively controlled by ppGpp?
High ppGpp causes depopulation of stringent promoters, resulting in high availability and redistribution of RNAP to relaxed promoters
Relaxed promoter = weak but resistant to ppGpp = upregulation of AA biosynthesis
What experiment showed that translation rates can alter transcription rates?
Compared rates in E. coli under normal and inhibited Examples:
- Chloramphenicol is translation elongation inhibitor
- Glycerol is poor C source, reducing growth rates and gene expression
Ratio between transcription and translation remained constant at 3nt/aa
Functional and physical interactions of RNAP and ribosome?
RNA synthesised by RNAP is template of ribosome
Translation elongation rates are dependent on transcription rates
Slow ribosomes can hold back elongating RNAPs due to coupling via physical interactions
What is transcription polarity?
ORFs downstream of a nonsense mutation (stop codon) will not be expressed
Naked mRNA will recruit rho and transcription will be terminated
What is transcription-translation coupling?
Yield of transcription meets need of translation
Coupled via physical interactions
What is the structural basis of TTC?
In cellulo cross-linking of RNAP-ribosome complexes revealed NusG and NusA transcription elongation factors
Chemical cross-linking using ChIP-Seq
Peptide and interaction identification with mass spec
Mapping cross-linked peptides onto structure
Ab initio structural modelling possible
Advanced structural reviews: showed that addition of factors including NusG leads to coupling of molecular machines
What is the role of NusG and NusA in TTC?
NusG bridges RNAP and 30S ribosome physically
NusA coordinates elongation and termination, stabilising interaction between RNAP and ribosomes
What is the RNAP response to translation inhibitors that mimic AA starvation in archaea?
Synthetases inhibited so loading of tRNAs prevented
Translation inhibition redistributes RNAP genome wide within 5 mins
Reduces RNAP EI (escape index) on protein coding genes indicative of TTC
EI remains unaffected on non-coding genes (rRNA etc.)
Experiments investigating transcription polarity of Sulfolobus?
Reporter gene assay:
Contain fluor protein downstream so fluor proprotional to level of expression
Interferes with stop codons at specific position in mutlicistronic operons
Introducing stop codon results in significant decrease in reporter gene activity (lacS)
Northern blot:
Large decrease in length of mRNA reporter gene (1.8 kb WT rpl3p -> 0.4 kb stop codon rpl3p)
Conclusion for upstream ORF with stop codon:
Represses downstream reporter gene (lacS) expression and activity
Generates truncated mRNA by premature degradation
- Ribosome functions as antiterminator
What is the mechanistic basis for elongation inhibition?
Mutual exclusive binding of aCPSF1 and ribosome to the Spt5-KOW domain of RNAP elongation complex
Balance between premature termination and antitermination adjusts promoter strength
Responses to environment are fed into transcription machinery
