(low muscle tone, short, incomplete sexual development, hyperphagia)
Rho factor needs
50-90nt region with high C content
3 drugs to stop RNA transcription
Actinomycin D (intercalates), Rifamycin (binds B subunit), a-amanatin (interferes with translocation)
Label with 32P. Add binding protein. Limited nuclease digestion with DNAse 1. Visualise using autoradiography.
How do we know most promoters are nucleosome free?
DNAse1 digestion occurs fastest in actively transcribed DNA
3 ways to alter histones
Methylation: 3 to lysine - more bulky: REPRESSES TRANSCRIPTION
Acetylation to lysine: less positive charge
Phosphorylation of serine: 2 negative charges
5'cap, 3 tail
how is eukaryotic transcription stopped
Pol II carries on for ages. pre-mRNA is cleaved between AAUAAA and GU/U rich sequence.
TF. Recognises GC/TC rich regions.
2 domains: N: Pro + Glu rich activation domain
C: DNA binding, 4 Zn fingers
Normal cells: 4 alternatively spliced variants. truncated forms lack domains.
hydrolytic deaminations (C->U or A->I)
Inosin can bind to U, C or A
When does large subunit bind?
Initiating AUG codon
Distinguishing between AUG codons
Eukaryote: first one along from 5'cap
Bactera: Shine Dalgarno sequence (GGAGG) recognised by AACAA in 16S rRNA of 30S subunit
Non-cap dependent translation initiation
Internal initiation of translation using IRES (internal ribosomal entry site). Found using dicistronic assays: assayable enzymes. In picornaviruses (polio, hep c). Long 5' UTR. Hep C IRES major drug target.
Met-tRNA to P site, via eIF2 (eukaryotic initiation factor) (interferon response, kinase)
All AA-tRNA to A site, via GTP and EFTu (EEF1)
Peptide bond by transfer of peptide from P to A site, via peptidyl transferase activity of large ribosomal subunit (ribozyme) - no energy use.
Translocation, via GTP and EFG (EEF2)
DNA sequences, bind small molecules. eg thiamine.
KFERQ: lots from liver, kidney during fasting.
E1, E2 (to SH) (activating, conjugating). UBIQUITIN LIGASE: to lysine, isopeptide bond. D box in cyclins required?