RNA is a single strand molecule composed of subunits called nucleotides joined by phosphodiester bonds.

Transcription

1 The Versatility of RNA

2 Primary structure of RNA RNA is a single strand molecule composed of subunits called nucleotides joined by phosphodiester bonds. Each nucleotide subunit is composed of a ribose sugar, a phosphate group, and a nitrogenous base. The common bases in RNA are adenine (A), guanine (G), cytosine (C), and uracil (U). PYRIMIDINES PURINES

3 Six major types of RNA RNA is involved in a wide range of cellular processes along the pathways of gene expression, including DNA replication, RNA processing, mrna turnover, protein synthesis. Some of the major types of RNA: Ribosomal RNA (rrna) is an essential component of the ribosome. Messenger RNA (mrna) is a copy of the genomic DNA sequence that encodes a gene product and binds to ribosomes in the cytoplasm. Transfer RNA (trna) is a small RNA charged with a specific amino acid. It delivers to the ribosome the appropriate amino acid.

4 Six major types of RNA Small nuclear RNA (snrna): plays a role in pre-mrna splicing, a process which prepares the mrna for translation. Small nucleolar RNA (snorna): plays a role in RNA processing. MicroRNA (mirna): involved in post-transcriptional gene regulation; each mirna binds to complementary sequence in a target mrna, usually resulting in gene silencing, by triggering degradation of mrna or by blocking translation by the ribosome

5 Secondary structure of RNA

6 Secondary structure of RNA Secondary structure: single-stranded RNA molecules fold into a variety of secondary structural motifs that are stabilized by both Watson- Crick and unconventional base pairing. Helix formation occurs within one single-stranded chain of nucleotides. The binding of divalent metal ions (such as Mg 2+, Ca 2+, etc) to the RNA is often critical to the formation of a stable, folded conformation because this ions can shield the negative charge of the RNA backbone, allowing regions of the molecule to pack more closely together.

7 Motifs include: Base-paired stems helix (with non canonical base pairs); Hairpin (U-shaped) loops; Internal loops Bulges Junction

8 Base-paired RNA adopts an A-type double helix The 2 -hydroxyl group hinders formation of a B-type helix. A-type RNA helices (right-handed) with Watson Crick base pairs have a deep, narrow major groove that is not well suited for specific interactions with ligands. The minor groove is shallow and broad and includes the ribose 2 -OH groups which are good hydrogen bond acceptors. Because of these structural features, it is common for RNA to be recognized by RNA-binding proteins in the minor groove. Minor Groove Major Groove

9 RNA helices often contain non-canonical base pairs >20 different types of non-canonical (non-watson-crick) base pairs. Widen the major groove and make it more accessible to ligands or proteins. The most common of these are:

10 AGC and ACG base triples RNA structure also frequently involve unconventional base pairing such as base triples. Typically involve one standard base pair. The third base interacts in a variety of unconventional ways.

11 Do these unusual base interactions have any functional significance? Yes! Non canonical base pairs and base triples are important mediators of: RNA self-assembly. RNA-protein interactions. RNA-ligand interactions. (e.g., widen the major groove making it more accesible to ligande)

12 trna structure trna is transcribed as a molecule about twice as long as its final form. The pre-trna transcript is then cut at both the 5 and 3 ends. The average trna is about 76 nt long. All trnas fold into the same general shape. Cloverleaf secondary structure L-shaped tertiary structure

13 trna structure More than 50 modified bases in trna. Modifications include simple methylation to complete restructuring of the purine ring. For example: Methylation: substitution of an atom or group by a methyl group (CH 3 )

14 trna structure Inosine (I) was the first modified nucleoside to be identified in trna Pseudouridine (ψ) was the first identified in any RNA.

15 trna loops each have a separate function trna has the sequence ACC on the 3 to which the amino acid is attached T-loop Involved in recognition by the ribosome. D loop recognition by the aminoacyl trna synthetases that charge the appropriate amino acid Anticodon loop base pairs with the mrna codon

16 Tertiary structure of RNA

17 Common tertiary structure motifs in RNA PSEUDOKNOTS MOTIF A pseudoknot motif forms when a single-stranded loop base pairs with a complementary sequence outside this loop and folds into a three-dimensional structure by coaxial stacking. The RNA appears to be tied in a knot. For example, the RNA component of Telomerase has a highly conserved For example, the RNA component of Telomerase has a highly conserved pseudoknot that is essential for its function.

18 Common tertiary structure motifs in RNA TETRALOOP MOTIF A stem loop with the tetraloop sequence UUUU is particulary stable due to special base-stacking interaction in the loop RIBOSE ZIPPER MOTIF This motif stabilizes helix-helix interactions between the minor groove. The interactions involve hydrogen bonding between the 2'-OH of a ribose in one helix and the 2-oxygen of a pyrimidine base of the other helix.

19 Common tertiary structure motifs in RNA KISSING HAIRPIN LOOP Two hairpin loop forms a kissing interaction by base pairing between single-stranded regions of two hairpin loops with complementary sequences.

20 Two Coaxial stacked arms form the familiar L-shape of trna Coaxial stacking, or helical stacking, occurs when the nucleotide bases from two separate base-paired stems stack and align to form what appears as a continuous helix. 7 bp acceptor stem in trna stacks on the 5 bp T stem to form an A- type helical arm. The D stem and anticodon stem stack to form a second helical arm How do these complicated structure get folded properly in the cell???

21 Classic definition of an enzyme: Ribozymes An enzyme increases the rate, or velocity, of a chemical reaction without itself being changed in the overall process. Enzymes reduce the activation energy, E a, of a reaction. Activation energy, E a :minimum energy that must be input to a chemical system in order for a chemical reaction to occur.

22 Ribozymes RIBOZYMES RNA molecules with catalytic activity. Naturally occurring ribozymes can be autocatalytic, which lead to their own modification, or they can be true enzymes. Form substrate-binding sites. Lower the activation energy. Allow reaction to proceed much faster. Mode of ribozyme action Some ribozymes use a two-metal-ion mechanism for catalysis. (Binding of divalent cations (Mg 2+ ) in the active site is critical for their folding into an active state) Other ribozymes use general acid-base chemistry, by donating or accepting protons during chemical step of reaction.

23 Two different groups based on difference in size and reaction mechanism: Small ribozymes Naturally occurring ribozymes Vary in size from 40 to 154 nucleotides. Cleave RNA to generate a 2-3 -cyclic phosphate and a product with a 5 -OH terminus. This group of small ribozymes includes

24 The hammerhead, so called for its three helices in a T shape, is the most frequently found catalytic motif in plant phatogenic RNAs called virions.

25 Naturally occurring ribozymes Large ribozymes Vary in size from a few hundred to 3000 nucleotides. Cleave RNA to generate 3 -OH termini. RNA components of:

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27 Evidence for the RNA world hypothesis Life first existed in the form of replicating RNA molecules. RNA has all the structural prerequisites for self-replication. RNA genomes are widespread among viruses. RNA molecules: Self-fold into 3-D structures. Recognize other macromolecules and ligands with precision. Catalyze a diversity of reactions. The ribosome is a ribozyme. Compared with DNA or protein, RNA is clearly the most selfsufficient molecule. RNA molecules are capable of doing basically all that proteins can do.

28 Evidence for the RNA world hypothesis Later, during a hypothetical transitional period, RNA catalyzed the synthesis of proteins and these proteins catalyzed the transition from RNA to DNA.

29 The Discovery of Reverse Transcriptase

30 Howard Temin Studied RNA tumor viruses most of his life His early days he spent time working on Rous sarcoma virus (RSV)

31 Howard Temin Hypothesis The RNA genome of RSV is converted into a DNA provirus.

32 Howard Temin Hypothesis RSV is sensitive to inhibitors of DNA synthesis and suggesting that DNA, complementary to the RSV genomic RNA, is present in transformed cells.

33 David Baltimore Spent time studying virus replication He looked at RNA and DNA at a more biochemical view looking deep within individual virions He later focused on RNA tumor viruses and concluded with Rauscher murine leukemia virus (R-MLV)

34 Experiment They disrupted their pure stock viruses by using nonionic detergents Can a retrovirus perform DNA synthesis? Then radiolabeled deoxthymidine triphosphate (dttp) along with three deoxynucleotide triphosphates (datp, dctp, dgtp) to the virion preparation.

35 Did these reactions contain any incorporation of radioactive dttp into DNA? YES! Each experiment, dttp turned into nucleic acid

36 RNA secondary structure Pseudoknot Hairpinloop Dublehelix Internal loop Bulge loop Multibranch loop (helical junction)

37 Probes Target Detection DMS A(N1)>>C(N3) Primer extension CMCT U(N3)>>G(N1) Primer extension T1 RNase Unpaired G 5 -end and 3 -end labelling T2 RNase Unpaired A>C,U,G 5 -end and 3 -end labelling V1 RNase Paired or stacked N 5 -end and 3 -end labelling

38 ladder G C C T1 T2 V C C T1 T2 V C C T1 T2 V1

39 strong T1 and T2 cuts weak T1 and T2 cuts strong V1 cuts weak V1 cuts