Functional RNA - Introduction Biochemistry 4000 Dr. Ute Kothe

Reading Biochemistry, Voet, 3 rd edition –Chapter (Posttranscriptional Processing) & 32.2 (tRNAs) Reviews: –Wakeman et al., TIBS 2007 (riboswitches) –Edwards et al., Curr Opin Struct Biol 2007 (riboswitches) –Scott, Curr Opin Struct Biol 2007 (ribozymes) –Doudna & Lorsch, Nat Struct Mol Biol 2005 (ribozymes)

Functional RNA - Classes Ribosomal RNA tRNA Spliceosomal RNA (small nuclear RNAs = snuRNAs) Telomerase RNA RNA modification complexes: small nucleolar RNA = snoRNA Ribozymes Riboswitches microRNAs 4.5 S RNA (signal recognition particle) Etc.

Primary & Secondary Structure Yeast tRNA Phe Primary Structure: Sequence of nucleotides in (single-stranded RNA) Secondary Structure: Watson Crick base-pairing - can be predicted by computer algorithms e.g. tRNA cloverleaf structure

RNA helices Voet, Chapter A-RNA resemble A-DNA wider an flatter right- handed helix than B-DNA 11.0 bp per turn pitch: 30.9 Å base-pairs are inclined by 16.7º to the helix axis similar conformation is adopted by RNA-DNA hybrid

Secondary structure elements Bulge Hairpin (stem-loop) Pseudoknot

Tertiary Structure Yeast tRNA Phe 3D structure Stabilized by tertiary interactions: hydrogen bonds stacking interactions e.g. in tRNA tertiary base-pairs between D and T loop

Tertiary Interactions in tRNA

Non Watson-Crick Base-Pairs Hoogsteen base-pairs compared to Watson-Crick base-pairs If not constrained in a helix, basically every edge of the nucleobase can participate in base-pairing to another nucleobase.

RNA structural elements U turn Kissing Hairpins GNRA tetraloop K turn A minor motif

RNA Modifications about 100 different modifications known mainly base modification: pseudouridine most common methylation of 2’OH of ribose moiety individual pathway for each modification believed to stabilize RNA structure may modify base pairing (e.g. 5-oxyacetic acid in first anticodon position)

RNA World Hypothesis Evolution of life may have started with RNA as the first biomolecule since RNA can store information (such as DNA) and catalyze reactions (such as proteins). Limitations of RNA compared to proteins: Few functional groups Low k cat Low stability Evolution: RNA RibonucleoproteinsProteins RNPs

Ribozymes Catalytic activity of RNA: Peptide bond formation Phosphodiester cleavage RNA ligation Cyclic phosphate hydrolysis Limited polymerization of RNA RNA phosphorylation RNA aminoacylation Diels-Alder addition Glycosidic bond formation Natural Ribozymes Artificial Ribozymes -Generated by in vitro selection

Ribozymes cleaving RNA Hairpin Ribozyme Hammerhead ribozyme Hepatitis Delta Virus Ribozyme (HDV) Varkud satellite ribozyme glms ribozyme RNase P (group I introns) (group II introns) General Mechanism of Phosphodiester cleavage:

RNase A vs. HDV ribozyme RNase A: Acid-base catalysis by 2 His (for details see Voet) HDV ribozyme: Acid-base catalysis by Cytidine 75 Involvement of a Mg 2+ What is the advantage of His over nucleobase for acid-base catalysis?

In vitro selected RNAs 1.Aptamers – small RNAs binding specific ligands 2.Ribozymes – small RNAs catalyzing desired reactions Diels Alder Ribozyme  Usually less active than natural ribozymes (lower affinities, lower rate enhancements)  due to limited number of evolution cycles

Riboswitch – Regulators of Gene Expression Mainly found in prokaryotes, rarely in eukaryotes respond to various small molecules Control a large number of genes in 5’ untranslated region (5’ UTR)  Evolutionary old & simple control mechanism?

Regulation types activation or repression transcriptional using a terminator hairpin or translational by sequestering the Shine- Dalgarno sequence

Guanine and Adenine riboswitch Structurally & Functionally very similar Highly selective Different regulation: (activaiton vs. repression) of downstream genes

Structure of Adenine Riboswitch Adenine binds at 3 helix junction Helices Pi & P3 stack Loop 2 and Loop3 form tertiary interactions Binding pocket for Adenine: specificity through Watson-Crick bp with U75 hydrogen bonds also to sugar edge of adenine adenine deeply buried within the riboswitch

RNA thermometer riboswitch regulating heat shock proteins at low temperatures, Shine- Dalgarno (grey box) sequences is sequestered by noncanonical base- pairing unfold at elevated temperatures & release the Shine-Dalgarno sequence