Nucleic Acid Structure & Recognition Flashcards
What is in DNA/ what structure
right handed helix, alternating sugar phosphate backbone. B - DNA right handed ds helix w antiparallel. major groove + minor groove. bases joined by N - glycosidic bond.
what are the types of interaction determine stability of double helix
base pairing, base-pair stacking, electrostatic repulstion
how is base pairing specified? and what does h bonding provide
specifide by h bonding and provides stability and specificity
how does adjacent base stacking stabilise it
base pairs = pi electron rings by entropically favoured exclusion of water. stacking is maximised by propellor twist so not in same plane. stacking favours extended conform. optimal in purine-pyrimidine bp steps. TATA readily melted
what are the two ways electrostatic repulsion interact
neg charged repulsion from backbone phosphates. inter-strand: destabilise double helix - repulsion reduced by higher ionic strength in vitro. intra-strand: favours an extended conformation - countered by bound proteins for bending.
name 3 structual variations of B-form DNA
Twist: rotation per base pair, roll: opening along base-pair long axis, slide: displacement along base-pair long axis. high propellor twist limits degree of slide.
facts about DNA major minor grooves. - dimensions may vary
unequal grooves. 120/240 angle between glycosidic bonds in each base-pair (not rotationally symmetrical) base=pairs are displaced from helical axis. major = wide and shallow and info rich. TA (MADA), CG (DAA) methyl, acceptor, donor
differences between A, B and Z - form DNA?
major groove B-DNA: seq specific recognition. Helical axis passes through base-pairs. A DNA - helical axis in major groove, shorter broader, major groove deep and narrow, RNA is this. Z - DNA left handed helix. unclear physiological relevance.
3 main diff between RNA and DNA
bases, sugar (ribose is 2’-OH not 2’ deoxyribose) and RNA vulnerable to alkaline hydrolysis, strands (single vs double)
points about RNA structure
lack of complementary strand, high degree of structure, can have non-canonical pairs.
can be catalytic (ribosome, spliceosome) || small molecule ligands (riboswitches)
what are some structural motifs of RNA
Base triple (UAU) and triple helices . example Expression and Nuclear Retention Elements (ENE) non coding RNAs
Pseudoknots: base-pairing of a loop sequence w complementary sequence outside the stem enclosing the loop. stabilised by co-axial stacking of the 2 helices.
what are the 5 steps to investigate mechanisms of Gene expression
- Clone and sequence the gene (genomic or cDNA)
- develop assay system (in vivo or in vitro)
- identify cis-acting sequences
- identify trans-acting factors (protein or RNA) that bind cis-sequences
- how cis elements and trans factors combine to control function
when choose genomic or cDNA clone
investigate transcription or pre-mRNA processing = genomic clone
functions of mRNA (in vitro translation assay and ORG has not yet been generated by splicing) = genomic clone not as good
cDNAs from mRNAs. no promoters, introns but additional features like poly A tail. investigate mRNA functions like translation and stability. used to express eukaryotic proteins in e.coli. common to design synthetic genes.
what are in vivo or in vitro assays
in vivo = intact living cells/organisms. physiological conditions, difficult to monitor reaction intermediate, not much control not physio if test gene too abundant.
in vitro = test in cell extract. precise control, purify trans factors, often inefficient, unphysiological
