Secondary and tertiary structure of RNA

Secondary and tertiary structure of RNA

1. SECONDARY AND TERTIARY STRUCTURES OF RNA Presented by RAJWANTI SARAN Ph. D. 1 st Year (PBG) RARI (SKNAU), Durgapura , Jaipur

2. RNA (Ribonucleic Acid) A nucleic acid that carries the genetic message from DNA to ribosomes and is involved in the process of protein synthesis is referred to as RNA. Ribonucleic acid is one of the two types of nucleic acids found in all cells. Some viruses use RNA instead of DNA as their genetic material. Ex. TMV, MS2 & R17 phages and viroids . RNA like DNA is a polynucleotide. RNA is either single stranded (usually) or double stranded.

3. Basic structure of RNA Back bone is sugar and phosphate group Nitrogenous bases linked to sugar moiety project from the backbone Nitrogenous bases (A, U, G & C) are linked to pentose sugar through N- glycosidic linkage to form a nucleoside Phosphate group is linked with 3’OH of nucleoside through phosphoester linkage 2 nucleotides are linked through 3’-5’ phosphodiester linkage to form a dinucleotide .

4. Structure of RNA

5. Types of RNA Messenger RNA (mRNA ) Ribosomal RNA ( rRNA ) Transfer RNA ( tRNA ) Small Nuclear RNAs ( snRNAs ) Micro RNAs ( miRNAs ) Small Interfering RNAs ( siRNAs ) Guide RNA ( gRNA ) Complementary RNA( cRNA ) Negative sense RNA O ther types

6. Messenger RNA (mRNA) Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes , the protein synthesis factories in the cell It is coded so that every three nucleotides (a codon ) correspond to one amino acid In eukaryotic cells, once precursor mRNA ( hnRNA ) has been transcribed from DNA, it is processed to mature mRNA This removes its introns —non-coding sections of the pre-mRNA The mRNA is then exported from the nucleus to the cytoplasm , where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA

7. Ribosomal RNA ( rRNA ) Ribosomal RNA ( rRNA ) is the catalytic component of the ribosomes Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome The ribosome binds mRNA and carries out protein synthesis Several ribosomes may be attached to a single mRNA at any time. Nearly all the RNA found in a typical eukaryotic cell is rRNA .

8. Transfer RNA ( tRNA ) Transfer RNA ( tRNA ) is a small RNA, chain of about 80 nucleotides It transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding

9. Small Nuclear RNAs ( snRNAs ) Sn RNAs are involved in the process of splicing ( intron removal) of primary transcript to form mature mRNA . The Sn RNAs form complexes with proteins to form Ribonucleoprotein particles called snRNPs

10. Micro RNAs ( miRNAs ) microRNAs , short non-coding RNAs present in all living organisms, have been shown to regulate the expression of at least half of all human genes . These single-stranded RNAs exert their regulatory action by binding messenger RNAs and preventing their translation into proteins .

11. Small Interfering RNAs ( siRNAs ) Small interfering RNA ( siRNA ) are 20-25 nucleotide-long double-stranded RNA molecules that have a variety of roles in the cell. They are involved in the RNA interference ( RNAi ) pathway, where it interferes with the expression of a specific gene by hybridizing to its corresponding RNA sequence in the target mRNA. This then activates the degrading mRNA. Once the target mRNA is degraded, the mRNA cannot be translated into protein.

12. Guide RNA ( gRNA ) RNA genes that function in RNA editing, found in mitochondria by inserting or deleting stretches of uridylates (Us ). The gRNA forms part of editosome and contain sequences to hybridize to matching sequences in the mRNA to guide the mRNA modifications . Complementary RNA( cRNA ) V iral RNA that is transcribed from negative sense RNA and serves as a template for protein synthesis. Negative sense RNA V iral RNA with a base sequence complementary to that of mRNA during replication it serves as a template to the transcription of viral complementary RNA

13. RNA types and 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,

14. RNA Structure Organization The native structure of RNA molecules can be divided into three different levels of organization: Primary structure Secondary structure Tertiary structure.

15. Figure 3: (a)Primary structure (b) Secondary structure (c) Tertiary structure

16. Primary structure It denotes the ribo -nucleotide sequence (commonly referred to as base sequence) of the molecule. Usually, the base-sequence of an RNA molecule only consists of a combination of the bases A, G, C, U. Furthermore, modified bases such as pseudouracil ( ψ ) are represented by their most-similar standard base.

17. Secondary structure The secondary structure is formed by a subset of the cis -Watson-Crick/Watson-Crick base pairs contained in an RNA molecule. This includes the standard A-U and G-C pairings already known from the formation of DNA helices as well as the so-called G-U wobble-pairs. Successive base-pairs form energetically favourable and thus stable stem- regions. The unpaired regions between two stems are called loops. E.g. The typical secondary structure of a tRNA consists of a 3-multiloop with three outgoing hairpin loops. This secondary structure is commonly referred to as cloverleaf or butterfly.

18. The secondary structure of an RNA molecule is formed by a number of secondary structure segments (motifs).

19. Secondary structure motifs can be classified into following loop classes:

20. Secondary structure motifs

21. A bulge loop Bulge loops have unpaired bases on only one strand in a double-stranded region, whereas the other strand only has paired bases [5]. The size of the bulge loop is at least the size of one unpaired base, but in principle there is no upper limit [5]. They have the ability to bend a stem and thereby influence the three-dimensional structure.

22. An internal loop Internal loops have unpaired bases on both strands in a double-stranded region. The thermodynamical stability of the loops depends on the types and the number of the unpaired bases [5]. If the number of the unpaired bases in both strands are of equal size, the internal loop is called symmetric[5]. Nevertheless the loop can be very inflexible due to stacking and/or hydrogen bonds.

23. A multi loop Loops which connect more than two helices are called multi loops. In between the helices unpaired bases can be found. Together with the closing base pair, the unpaired bases are decisive for the stacking of the helices and thereby they form the three-dimensional structure [5]. Very often it can be observed that four helices are connected within a multi loop, for instance in tRNA , but also more or less helices can be connected [5].

24. A hairpin loop A hairpin loop describes the structure of a sequence that folds back on itself, usually a stem or a double helix and thereby forming an unpaired loop. Such a loop is called a hairpin loop and is formed relatively quick [5]. The time needed to grow the loop is at its minimum in the range of only a few microseconds and is growing with the length of the unpaired loop [5].

25. Continue… The thermodynamical stability of the loop depends on the sequence of the loop, on the type of the closing base-pair and on the size of the loop [5]. A hairpin loop needs at least four unpaired bases and often loops of five unpaired bases are the most stable ones [5]. Very stable tetraloop hairpins can be found in rRNA and even bigger hairpin loops can for instance be found in tRNA : The anticodon loops consist of seven bases [5].

26. A pseudoknot A pseudoknot is a tertiary structural element of RNA. It is formed by base-pairing between an already existing secondary structure loop and a free ending [5]. Nucleotides within a hairpin loop form base pairs with nucleotides outside the stem [7]. Hence base pairs occur that overlap each other in their sequence position. Fig. formation of a pseudoknot with coaxial stacking of the two helices

27. Figures of different types of loops

28. Tertiary structure Base-pairs that do not belong to the secondary structure together with pseudo-base-pairs form the tertiary structure of the molecule. This includes other atomic interactions such as vanderwaals forces, electrostatic and hydrophobic interactions and hydrogen-bonds between e.g. base and ribose residues. Tertiary contacts are interactions between distinct secondary structure elements. They induce local and/or global structure folds and as such are dominantly responsible for the overall three-dimensional structure of an RNA molecule [4].

29. Continue…. Tertiary interactions can occur between two helical motifs (stem-stem), between two unpaired (loop-loop), and between an unpaired region and a stem region (loop-stem) [4]. In the three-dimensional structure of a tRNA molecule, the stems of the D-loop and the T-loop, as well as the acceptor-stem and the stem of the anticodon -loop stack upon another (coaxial stacking stem-stem interaction). The typical L-shape of a tRNA molecule is yielded by the stacked stem regions as well as the kissing hairpin loop-loop interaction between the D-loop and T-loop hairpins.

30. Tertiary structure interactions Interactions Between Helical Motifs (stem-stem) Coaxial Stacking The Adenosine Platform 2'-Hydroxy-Mediated Helical Interactions 2. Interactions Between Helical and Unpaired Motifs (stem-loop) Base Triples and Triplexes The Tetraloop Motif The Metal-Core Motif The Ribose Zipper 3. Tertiary Interactions Between Unpaired Regions(loop-loop) Loop - Loop Interactions The Pseudoknot

31. Coaxial Stacking The most fundamental method by which RNA achieves higher order organization, is a consequence of the highly favorable energetic contributions of stacking interactions between the pie-electron system of the nucleotide bases to the overall stability of nucleic structure. The contribution of coaxial stacking to the global fold of an RNA was first observed in the crystal structure of tRNAPhe .[6, 8, 9] In the 3-D structure the stems of the D- and anticodon arms stack upon one another as do the stems of the T-arm and aminoacyl acceptor arm [9]. These two coaxial stacks are oriented perpendicularly with respect to one another by tertiary interactions between the D and T-loops to yield the overall L-shape of the molecule. The predominance of coaxial stacking in the organization of RNA structure is also evident in the structures of the P4-P6 domain and the hepatitis delta ribozyme .

32. Continue.. The organization of junctions, in which three or more helices intersect, by coaxial stacking is often achieved through the binding of divalent metals near the site of the stack. The direct influence of metal-ion binding on the folding of this secondary structural motif is clearly demonstrated in studies of the three-way junction at the catalytic center of the hammerhead ribozyme . In the crystal structure two of the helices are seen to coaxially stack, and the third is oriented relative to the coaxial stack by both tertiary contacts and hydrated magnesium ions specifically bound to the RNA.

33. Role of secondary and tertiary structures of RNA The different structures are important for catalytic, regulatory or structural roles within the cells. RNA secondary structure prediction has applications to the design of nucleic acid probes [10]. It is also used by molecular biologists to help predict conserved structural elements in non-coding regions of gene transcripts [10]. There is also an application in predicting structures that are conserved during evolution [10]. Tertiary structure prediction is important for understanding structure–function relationships for RNAs whose structures are unknown and for characterizing RNA states recalcitrant to direct analysis.

34. Conclusion Ribonucleic acids are negatively charged polymers assembled from four different types of monomers. Each monomer is made of an invariant phosphorylated sugar to which is attached one of the four standard nucleic acid bases; the pyrimidines uracil and cytosine, and the purines guanine and adenine. The first level of organization is thus the sequence of bases attached to the sugar–phosphate backbone. In salty water, the RNA molecules fold back on themselves via Watson–Crick base pairing between the bases (A with U, G with C or U) leading to double-stranded helices interrupted by single-stranded regions in internal loops or hairpin loops. The enumeration of the base-paired regions or helices constitutes a description of the second level of organization, the secondary structure.

35. Continue… Under appropriate conditions, structured RNA molecules undergo a transition to a three-dimensional (3D) fold in which the helices and the unpaired regions are precisely organized in space. This folding process usually depends on the presence of divalent ions, such as magnesium ions, and on the temperature. The tertiary structure is the level of organization relevant for biological function of structured RNA molecules.

36. References [1] Christine E. Hajdin1, Feng Ding2, Nikolay V. Dokholyan2 and Kevin M. Weeks1 2010. On the significance of an RNA tertiary structure prediction . RNA, 16: 1340-1349. [2] Christian Schudoma (3680 750) 2014. A Fragment Based Approach to RNA Threading. [3] Philip C. Bevilacqua,1,2,3 Laura E. Ritchey,1,3 Zhao Su,4 and Sarah M. Assmann4 2016 . Genome-Wide Analysis of RNA Secondary Structure. Annu. Rev. Genet.. 50:235–66. [4] Batey , R. T. and Rambo, R. B. and Doudna , J. A. 1999. Tertiary Motifs in RNA Structure and Folding. Angew . Chem. Int. Ed., 38:2326–2343. [5] Steger G. 2003 . "Bioinformatik- Methoden zur Vorhersage von RNA- und Proteinstrukturen", Birkh auser Verlag . [6] S. H. Kim, F. L. Suddath , G. J. Quigley, A. McPherson, J. L. Sussman , A. Wang, N. C. Seeman , A. Rich 1974. Science, 185, 435 ± 440. [7]Rivas E., Eddy S.R. 1999."A Dynamic Programming Algorithm for RNA StructurePrediction Including Pseudoknots ", Academic Press. [8] J. D. Robertus , J. E. Ladner , J. T. Finch, D. Rhodes, R. D. Brown, B. F. C. Clark, A. Klug 1974. Nature, 250, 546 ± 551. [9] A. Jack, J. E. Lander, A. Klug 1976. J. Mol. Biol., 108, 619 ± 649. [10] ESI Special Topic, "Fast Breaking Comments by Michael Zuker",http : m== www:esi topics:com = fbp =2004=august04 MichaelZuker:html,16.08.2008

37. THANK YOU