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Chapter 5The Structure and Versatility of RNA

Includes:1. RNA contains ribose, uracil and is usually single-

stranded

2. RNA folds back to form local regions of double helix

3. RNA can fold up into complex tertiary structures

4. Directed evolution selects RNA that binds small molecules

5. Some RNAs are enzymes

RNA structure is varied

• RNA is single stranded

• Diverse tertiary structures

Intrastrand base pairing

Nonclassical base pairing

Base-backbone interaction

Knot-like configuration• Some RNAs are enzymes

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Figure 5-1 structural features of RNA

1. RNA: ribose, uracil, usually single-stranded

Figure 5-2 U pairs with A

Functions of RNA• mRNA: an intermediate between gene

and the protein synthesizing-machinery

• tRNA: an adaptor, between the codons in mRNA and amino acids

• rRNA: structural roles in ribosomes

• siRNA: gene expression regulator

• Ribozymes: enzymes that catalyze essential reaction in the cell

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2. RNA folds back to form local regions of double helix

Figure 5-3 Double helical characteristics of RNA

Stem-loop

http://nar.oxfordjournals.org/content/38/2/683/F1.small.gif

A stem-loop with the “tetraloop” sequence (UUCG) is unexpectedly stable. This presents an important model system for the study of RNA structure and dynamics at the molecular level.

Base-stacking interaction

Figure 5-4 Tetraloop

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PseudoknotFormed by base pairing between noncontiguous complementary sequences

Figure 5-5 Pseudoknot

non-Watson-Crick base pair

Figure 5-6 G:U base pair

GC, AU, GU, GA for RNA base pairing enhances capacity for self-complementarity and help to form local regions of double helix, but not the long-range, regular helicity of DNA.

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major

dsRNA adopts A-form structureRNA: 2‘-OH The major groove is too small

to be accessed by amino acid side chain for interaction. So, dsRNA is not suited for sequence-specific interaction with protein. However, some proteins bind to RNA in a sequence-specific manner, by recognizing hairpin loops, bulges, and distortions caused by noncanonical base pairs. For example, tRNA synthetases with their tRNA

Biological functions of RNA secondary structures

PrfA, a thermosensor gene in Listeria monocytogenes, is a transcription factor that control the expression of virulence genes

Figure 5-7. The prfA regulatory gene is controlled at translation level by temperature-dependent unmasking of ribosome-binding site (RBS)

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3. RNA can fold up into complex tertiary structures• Without the constraints imposed by forming

long, regular helices, RNA has great rotational freedom in the backbone of its non-base-paired region.

• Various kinds of base interaction: (unconventional, triple) base pairing + base-backbone

• Proteins help the formation of 3o structures by large RNA by shielding the negative charge of phosphates.

• The RNA 3o structures are not necessarily static, and may exist in one or more alternative conformation, which might be related to its function.

Tertiary structure of the hammerhead ribozyme

Unusual triple base paring

Figure 5-8 U:A:U base triple

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An RNA switch controls protein synthesis by murine leukemia virus

Glutamine

Box 5-1 Figure 1. An equilibrium between two pseudoknot conformations controls translation through a stop codon

Inactive pseudoknot

Active pseudoknot, by H+

4. Directed evolution selects RNAs that bind small molecules

The structural complexity of RNA can be used to generate novel RNA species that have specific desirable properties by a process of directed evolution known as SELEX

SELEX: systemic evolution of ligands by exponential enrichment

Ligands: DNA or RNA ligands that bind to protein

Molecules bound by RNA aptamer: ATP, kanamycin, neomycin …

Figure 5-10 cycle for creating RNAs that bind small molecules by SELEX

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http://www.molmed.uni-luebeck.de/T.%20Restle/selex.html

Creating an RNA mimetic of the GFP by directed evolution

BOX 5-2 Figure 1. RNA-fluorphore complexes exhibiting a range of different colors.

BOX 5-2 Figure 2. Using SELEX to create metabolite sensors. The sensor contains bonding domains for a fluorophore (green) and a metabolite (purple)

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Green Fluorescence Protein

4-hydroxybenzlidene imidazolinone (fluorphore)

http://www.rcsb.org/pdb/101/motm.do?momID=42

5. Some RNAs are enzymesRibozyme: with features similar to the protein enzymes, such as active site, substrate binding site, cofactor binding site, etc.

• Such as RNase P that generates tRNA from a larger precursor.• Other ribozymes are involved in RNA splicing to remove

intron.

Proteins shield the negative charge on the RNA so that it can bind to the negatively charged substrate RNA.

www.science.ca/images/altman_rnase.jpg

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Figure 5-12. Structure of RNase P. Purple: RNA; Blue: protein; Yellow: tRNA

Figure 5-11. RNase P cleaves the 5’ end of a precursor to generate tRNA.

Leader segment

Catalytic center

Leader

The hammerhead ribozymeA sequence-specific RNase that is present in viroids, infectious RNA agents of plants

www.micro.siu.edu/micr302/miscfigsclark.html

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The hammerhead ribozyme cleaves RNA at cytosine 17 position

Red: Watons-Crick base pairBlue: substrate strandX17: Cytosine

Figure 5-13. Structure of the hammerhead ribozyme. (Left) A cartoon of the secondary structure with the three stems. (Right) 3D structure with magnesium (Red) in the catalytic center nearing the cleavage site. The colors correspond to each other.

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The hammerhead ribozyme cleaves RNA by the formation of a 2’,3’ cyclic phosphate

Mg2+

A ribozyme at the heart of the ribosome acts on a carbon center

RNase P and hammerhead both act on Phosphate (RNA), the ribozyme (peptidyl transferase) in ribosome act on C (amino acid).

The discovery of peptidyl transferase is a support for the hypothesis that contemporary, protein-based life arose from an early RNA world.

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Did life evolve from an RNA world?

Evidence: Peptidyl transferase is an RNA

RNA functions as genetic material and enzymes