2. Eukaryotic vs. Prokaryotic Transcription In eukaryotes, transcription and translation occur in separate compartments. In bacteria, mRNA is polycistronic; in eukaryotes, mRNA is usually monocistronic. –Polycistronic: one mRNA codes for more than one polypeptide –moncistronic: one mRNA codes for only one polypeptide 3 RNA polymerases in euk., 1 in prok. “Processing” of mRNA in eukaryotes, no processing in prokaryotes

3. Four different types of RNA, each encoded by different genes: 1. mRNAMessenger RNA, encodes the amino acid sequence of a polypeptide 2. tRNATransfer RNA, transports amino acids to ribosomes during translation 3. rRNARibosomal RNA, forms complexes called ribosomes with protein, the structure on which mRNA is translated 4. snRNASmall nuclear RNA, forms complexes with proteins used in eukaryotic RNA processing

5. Potential Steps for Regulation of Eukaryotic Gene Expression

7. CAPPING

8. Cap structure on the 5’ end of mRNA molecules

9. Cap Functions Cap provides: 1.Protection from some ribonucleases 2.Enhanced translation 3.Enhanced transport from nucleus 4.Enhances splicing of first intron for some mRNAs

10. AdoMet = S-adenosylmethionine, the methyl donor Capping: order of events and enzymes Product is Cap 1 (another Met at 3 rd nucl =>Cap2) or “RNA triphosphatase”

11. Capping occurs co-transcriptionally shortly after initiation guanylyltransferase (nuclear) transfers G residue to 5’ end methyltransferases (nuclear and cytoplasmic) add methyl groups to 5’ terminal G and at two 2’ ribose positions on the next two nucleotides pppNpN mGpppNmpNm

12. SPLICING

13. Overview of the processing of a eukaryotic mRNA

14. 1 2 3 4 5 6 7

15. Eukaryotes –The occurrence of introns varies. The majority of genes in vertebrates contain introns. –genes encoding histones do not have introns. The yeast have many genes that lack introns Prokaryotes –Most prokaryotes do not have introns. A few bacteria and archaebacteria have introns.

16. Splice Sites Conserved splice sites are shared by both the exon and the intron. Different sequences on the donor site (3’) and on the acceptor site (5’).

19. POLIADENYLATION

20. 3’ poly(A) tail Cleavage signal sequence

21. rRNA processing

22. Ribosome Structure S = Svedberg, a measure of sedimentation in centrifuge

23. Transcription – rRNA Processing

24. Ribosomal RNA Genes Tandemly repeated Non-transcribed spacers 45S rRNA precursor → 18S, 5.8S, 28S rRNA

26. Processing of Human 45S rRNA Precursor

27. Processing Bacterial rRNA Precursor

28. bacteria

29. vertebrates

30. RNA modification: snoRNA Small nucleolar RNA (snoRNA) has many modifying functions including methylation and pseudouridylation of pre-rRNA. The exact purpose of these modifications are still unknown except to say that they somehow guide the rRNA subunits to form a functional ribosome.

31. Transcription – rRNA Processing –Role of small nucleolar RNAs (snoRNAs) Packaged with proteins to form small nucleolar ribonucleoproteins (snoRNPs) snoRNPS associate with rRNA before it is fully transcribed Two groups of snoRNAs –U3 & antisense Antisense forms an RNA duplex –Recognition site for enzymes which modify pre-RNA

34. tRNA processing

35. Synthesis of tRNA: 1.tRNA genes also occur in repeated copies throughout the genome, and may contain introns. 2.Each tRNA (75-90 nt in length) has a different sequence that binds a different amino acid. 3.Many tRNAs undergo extensive post-transcription modification, especially those in the mitochondria and chloroplast. 4.tRNAs form clover-leaf structures, with complementary base- pairing between regions to form four stems and loops. 5.Loop #2 contains the anti-codon, which recognizes mRNA codons during translation. 6.Same general mechanism using RNA polymerase III, promoters, unique TFs, plus posttranscriptional modification from pre-tRNA.

36. Ribozyme

37. Sphere = cleavage point RNase P RNA forms a pocket around the pre-tRNA, creating an enzymaticly favorable environment for cleavage. Ribonuclease P (RNase P) is a holoenzyme that cleaves the 5¢ leader element of pre-tRNA to produce mature tRNA. It is found in all domains of life as well as mitochondria and chloroplasts. RNase P is made of a catalytically active ncRNA component and protein component.

39. Function of Unusual Bases Created post-transcriptionally. Purpose is sometimes to allow for promiscuous base- pairing: Inosine in the 1st “wobble” position of anticodon can bind to 3rd U, C or A in codon. This means that fewer different tRNAs are required. Others play a structural role.

41. Ribozymes Discovered in 1980’s (Cech & Altman, Nobel Prize 1989) RNA can act as an Enzyme and catalyse Reactions including Its own replication The RNA WORLD

42. Enzymes function to reduce the activation energy Reactions catalyzed by RNA can be characterized in the same way as classical protein enzyme reactions (Michaelis-Menten kinetics: 10 3 -10 6 )

45. Catalytic Mechanism of Group I Intron: Tom Cech et al.

46. Group II introns: found in bacteria and organellar genes of eukaryotic cells 1.Nucleophilic attack by the 2’-OH of an adenosine within the branch site 2.Conformational change releases the intron lariat and the mature exon Group II introns encode proteins involved in splicing, integration and RT, promoting intron mobility

47. Hammerhead Ribozyme Scott WG, Finch JT, Klug A. (1995) The crystal structure of an all-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81, 991-1002. Hammerhead ribozymes are catalytic RNAs found in plants and some pathogens. Their reactions are very limited, typically strand cleavage. They are all metalloenzymes, usually using Mg. Several hammerhead structures have been solved. This one is a minimized RNA which still retains catalytic activity; it has a 16 base “enzyme” strand and a 25 base substrate strand. In the crystal, however, the usual cleavage site at C17, has been replaced with a non-hydrolyzable 2’ deoxy nucleotide. The structure shows a “gamma” shaped molecule, with stems I, II, and III flanking a conserved 16-base core which is required for structure and activity. These core bases do not form Watson-Crick pairs, but a variety of adventitious interactions.

48. Ribonuclease P: found in all cells Site specific hydrolysis of tRNA, 5S rRNA and signal recognition particle RNA Two domain structure –Substrate recognition –Ribozyme active site Structural predictions of the RNA are made by doing phylogenetic comparisons

49. Hairpin ribozymes: plant virus satellite RNA’s mediate rolling circle replication Two main helical regions G8 is essential but its role in catalysis may be structural (this may be true for metal ions as well) G8 Active A ??

50. Hepatitis delta virus: is an RNA satellite virus of hepatitis B virus (HBV). HDV is the only catalytic RNA known to be required for the viability of a human pathogen and is the fastest known naturally occurring self-cleaving RNA (first-order rate of 52 reactions/min) Nested double pseudoknot in which the active site is buried (100-fold faster the Hammerheads) 2.3 Å resolution

51. The RIBOSOME is a RIBOZYME 5S rRNA 23S rRNA A-site tRNA P-site tRNA P A Adenine is highly conserved, participates in general acid- base catalysis to deprotonate the amine Ribosomal proteins act as scaffolding to orient the catalytic RNA RNA WORLD?

52. Emerging clinical applications of Ribozymes Emerging clinical applications of Ribozymes an alternative or in combination with siRNA 1.Gene inhibitors 2.Gene amenders 3.Protein inhibitors 4.Immunostimulatory RNA’s 4 main categories

53. Clinical applications for trans-cleaving ribozymes 1.Transduced cells are well tolerated and persist in the patient 2. Transduced cells may possess transient survival advantage over control cells Results Shortcomings 1.Long term efficacy is still a question. 2.Efficient gene delivery system. Chem. hammerhead RIBOZYME clinical trials in progress: anti-cancer (VEGF) and anti-viral (HIV)

54. Trans-splicing mediated repair of mutant transcripts The concept Exploit group I introns to cut and paste exons in a directed fashion A. The ribozyme is linked to a W.T. gene fragment B. Corrupt cis-splicing by inserting a W.T. exon fix p53 in cancer cells? SMaRT slpiceosome-mediated RNA trans-splicing (CFTR^F508)

56. Some modern day coenzymes may be the evolutionary remnants of modified nucleotides in catalytic RNA

57. Three successive stages in the evolution of a self- replicating system of RNA molecules capable of directing protein synthesis.