RNA Tertiary Structure

Additional Motifs of Tertiary Structure

• Coaxial helix• A minor motif• Pseudoknots• Tetraloops• Loop-loop• Ribose zipper• Kink turn motif

Coaxial helix

• Two separate helical regions stack to form coaxial helices as a pseudo-continuous (quasi-continuous) helix.

• Coaxial helices are highly stabilizing tertiary interactions and are seen in several large RNA structures, including tRNA, pseudoknots, the group I intron P4-P6 domain, and in the Hepatitis Delta Virus ribozyme.

A-minor motif

• The A-minor motif involves the insertion of minor groove edges of adenines into the minor groove of neighboring helices.

• It has four subtypes depending on the position of the adenine to the interacting Watson-Crick base pair.

ype 0: The N3 of the A (or other) residue is outside the O2' of the far strand of the receptor helix. Type I: The O2' and N3 atoms of the A residue are inside the minor groove of the receptor helix. The inserted base for the Type I interaction must be an adenine. Type II: The O2' of the A residue is outside the near strand O2' of the helix and the N3 of the A residue is inside the minor groove. The inserted base for the Type II interaction must be an adenine. Type III: The O2' and N3 of the A (or other) residue are outside the near strand O2' of the receptor helix.

Ribose zipper

• The ribose zipper is a tertiary interaction formed by consecutive hydrogen-bonding between the backbone ribose 2 -′hydroxyls from two regions of the chain interacting in an anti-parallel manner.

Pseudoknot

• When bases pair between nucleotides loops (hairpin or internal) and bases outside the enclosing loop, they form a pseudoknot.

• This structure often contains coaxial helices. • It can be a very stable tertiary interaction.

Loop-loop receptor

• The tetraloop-tetraloop receptor was identified by comparative sequence analysis.

• This tertiary interaction is characterized by specific hydrogen-bonding interactions between a tetraloop and a 11-nucleotide internal loop/helical region that forms the receptor.

• Other kinds of loop and receptor interactions, such as penta-loop/receptor and hexa-loop/receptor, are observed so this motif is call loop-loop receptor.

tRNA D-loop;T-loop

• The D-loop in tRNA contains the modified nucleotide dihydrouridine.

• It is composed of 7 to 11 bases and is closed by a Watson Crick base pair. The TψC-loop (generally called the T-loop) contains thymine, a base usually found in DNA and pseudouracil (ψ).

• The D-loop and T-loop form a tertiary interaction in tRNA.

Kissing hairpin

• The kissing hairpin complex is a tertiary interaction formed by base pairing between the single-stranded residues of two hairpin loops with complementary sequences

A new concept:the Ribozyme - enzymic RNA

• Exactly following the definition of an enzyme, the L-19 IVS RNA

• accelerates the reaction by a factor of around .

• is regenerated after each reaction each enzyme molecule can react with many substrate molecules.

1010

Ribozymes - Therapeutic Applications

• Simple structure, site-specific cleavage activity and catalytic capability, make ribozymes effective modulators of gene expression.

• Ribozyme-mediated gene modulation can target cancer cells, foreign genes that cause infectious diseases as well as other target sites (current research), and thereby alter the cellular pathology.

proteins

23S rRNA

peptidyl transfer reaction: P-site tRNA

5S rRNA

A-site tRNA

Ninety eight percent of the human genome

does not code for protein. What is its

function?

How much of human transcribed RNAresults in proteins?

• Of all RNA, transcribed in higher eukaryotes, 98% are never translated into proteins.

• Of those 98%, about 50-70% are introns • 4% of total RNA is made of coding RNA• The rest originate from non-protein genes, including rRNA,

tRNA and a vast number of other non-coding RNAs (ncRNAs)• Even introns have been shown to contain ncRNAs, for

example snoRNAs• It is thought that there might be order of 10,000 different

ncRNAs in mammalian genome

22

RNA functions

• Storage/transfer of genetic information

• Structural

• Catalytic

• Regulatory

D Dobbs ISU - BCB 444/544X: RNA Structure & Function

23

RNA functions

Storage/transfer of genetic information• Genomes

• many viruses have RNA genomessingle-stranded (ssRNA)

e.g., retroviruses (HIV)double-stranded (dsRNA)

• Transfer of genetic information • mRNA = "coding RNA" - encodes proteins

D Dobbs ISU - BCB 444/544X: RNA Structure & Function

24

RNA functions

Structural • e.g., rRNA, which is major structural component of

ribosomes BUT - its role is not just structural, also:

Catalytic RNA in ribosome has peptidyltransferase activity

• Enzymatic activity responsible for peptide bond formation between amino acids in growing peptide chain

• Also, many small RNAs are enzymes "ribozymes”

25

RNA functions

Regulatory Recently discovered important new roles for RNAs In normal cells:

• in "defense" - esp. in plants• in normal development

e.g., siRNAs, miRNAAs tools:

• for gene therapy or to modify gene expression• RNA aptamers

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 26

RNA types & functions Types of RNAs Primary Function(s)

mRNA - messenger translation (protein synthesis) regulatory

rRNA - ribosomal translation (protein synthesis) <catalytic>

t-RNA - transfer translation (protein synthesis)

hnRNA - heterogeneous nuclear

precursors & intermediates of mature mRNAs & other RNAs

scRNA - small cytoplasmic signal recognition particle (SRP)tRNA processing <catalytic>

snRNA - small nuclear snoRNA - small nucleolar

mRNA processing, poly A addition <catalytic>rRNA processing/maturation/methylation

regulatory RNAs (siRNA, miRNA, etc.)

regulation of transcription and translation, other??

RNA

mRNA ncRNA(non-coding RNA) Transcribed RNA with a structural,

functional or catalytic role

rRNARibosomal

RNAParticipate inprotein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall

nuclear RNA-Incl. RNA that

form part of the

spliceosome

snoRNASmall

nucleolar RNAFound in

nucleolus,involved in

modificationof rRNA

miRNAMicro RNA

Small RNA involved

regulation of expression

OthersIncluding large

RNAwith roles inchromotin

structure andimprinting

stRNASmall temporal RNA.

RNA with a role inDevelopmental timing.

siRNASmall interfering RNAActive molecules in

RNA interference

Small Nuclear RNAs

• One important subcategory of small regulatory RNAs consists of the molecules know n as small nuclear RNAs (snRNAs).

• These molecules play a critical role in gene regulation by w ay of RNA splicing.

• snRNAs are found in the nucleus and are typically tightly bound to proteins in complexes called snRNPs (small nuclear ribonucleoproteins, sometimes pronounced "snurps").

• The most abundant of these molecules are the U1, U2, U5, and U4/U6 particles, w hich are involved in splicing pre-mRNA to give rise to mature mRNA

MicroRNAs• RNAs that are approximately 22 to 26 nucleotides in length. • The existence of miRNAs and their functions in gene regulation w ere

initially discovered in the nematode C. Elegans .• Have also been found in many other species, including flies, mice, and

humans. Several hundred miRNAs have been identified thus far, and many more may exist.

• miRNAs have been show n to inhibit gene expression by repressing translation.

• For example, the miRNAs encoded by C. elegans, lin-4 and let-7, bind to the 3' untranslated region of their target mRNAs, preventing functional proteins from being produced during certain stages of larval development.

• Additional studies indicate that miRNAs also play significant roles in cancer and other diseases. For example, the species miR-155 is enriched in B cells derived from Burkitt's lymphoma, and its sequence also correlates w ith a know n chromosomal translocation (exchange of DNA between chromosomes).

Small Interfering RNAs

• Although these molecules are only 21 to 25 base pairs in length, they also work to inhibit gene expression.

• siRNAs were first defined by their participation in RNA interference (RNAi). They may have evolved as a defense mechanism against double-stranded RNA viruses.

• siRNAs are derived from longer transcripts in a process similar to that by which miRNAs are derived, and processing of both types of RNA involves the same enzyme, Dicer .

• The two classes appear to be distinguished by their mechanisms of repression, but exceptions have been found in which siRNAs exhibit behavior more typical of miRNAs, and vice versa.

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 32

miRNA Challenges for Computational Biology

• Find the genes encoding microRNAs

• Predict their regulatory targets

• Integrate miRNAs into gene regulatory pathways & networks

Computational Prediction of MicroRNA Genes & Targets

C Burge 2005

Need to modify traditional paradigm of "transcriptional control" by protein-DNA interactions to include miRNA regulatory mechanisms

D Dobbs ISU - BCB 444/544X: RNA Structure & Function 33

C. elegans lin-4 Small Regulatory RNA

We now know that there are hundreds of microRNA genes

(Ambros, Bartel, Carrington, Ruvkun, Tuschl, others)

lin-4 precursor

lin-4 RNA

“Translational repression”

V. Ambros lablin-4 RNA

target mRNA

C Burge 2005