Review from last time

1. Review from last time • Gene duplication occurs much more often than genome duplication • Gene duplication can provide a source of variation for the development of new functions in organisms • Transposable elements are interspersed sequences in all eukaryotic genomes • Be familiar with the structure and mobilization mechanisms for class 1 and class 2 elements • Be able to describe the potential impacts of mobile elements on a genome • The most current estimate is ~25-30,000 genes in our genome • Comparative genomics can provide information on the similarities and differences among genome and indicate what parts are ‘important’

2. Chapter 11:Gene Expression: From Transcription to Translation

3. This Chapter in One Slide details details details details details details details details details details details details details details details details details details details

4. Gene Expression • RNA – Ribonucleic acid • Slightly different from DNA • Uracil instead of Thymine • RNA is critical to all gene expression • mRNA – messenger RNA; created from a DNA template during transcription • tRNA – transfer RNA; carriers of amino acids; utilized during translation • rRNA – ribosomal RNA; the site of translation • Other RNAs – snoRNA, snRNA, miRNA, siRNA • Many RNAs fold into complex secondary structures

6. Transcription • Transcription – the process of copying a DNA template into an RNA strand • Accomplished via DNA dependent RNA polymerase (aka RNA polymerase)

7. Transcription • By the end of this series of slides, you should be able to explain much of this animation • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf

8. Transcription • Begins with the association of the RNA polymerase with the DNA template • Which brings up DNA protein interactions • Some enzymes have evolved to recognize specific DNA sequences • One such DNA sequence is called a promoter • The promoter is the assembly point for the transcription complex • RNA polymerases cannot recognize promoters on their own, but require the help of other proteins (transcription factors)

9. Bacterial RNA polymerase can incorporate 50 - 100 nucleotides/sec • Most genes in cell are transcribed simultaneously by numerous polymerases • Polymerase moves along DNA in 3' —> 5' direction • Complementary RNA constructed in 5' —> 3' direction • RNAn + NPPP —> RNAn+1 + PPi

10. Transcription • Prokaryotic Transcription • One type of RNA polymerase with 5 subunits tightly associated to form core enzyme • Core enzyme minus sigma (σ) factor will bind to any DNA. • By adding σ, RNA pol will bind specifically to promoters

11. Transcription • Prokaryotic Transcription • Bacterial promoters are located just upstream of the RNA synthesis initiation site • The nucleotide at which transcription is initiated is called +1; the preceding nucleotide is –1 • DNA preceding initiation site (toward template 3' end) are said to be upstream • DNA succeeding initiation site (toward template 5' end) are said to be downstream

12. Transcription • Prokaryotic Transcription • Similar DNA sequences are seen associated with genes in roughly the same location for multiple genes in bacteria • The consensus sequence is the most common version of such a conserved DNA sequence • DNA sequences just upstream from a large number of bacterial genes have 2 short stretches of DNA that are similar from one gene to another (-35 region & -10 region) • T78T82G68A58C52A54 -- 162117521819 -- T82A89T52A59A49T89 • - 35 region spacer -10 region • σ factors and polymerases recognize the sequences and bind to them • TTGATA • TTGACA • CTGACG

13. Transcription • Eukaryotic Transcription • Three distinct RNA polymerases, each responsible for synthesizing a different group of RNAs • RNA polymerase I (RNA pol I) - synthesizes the larger rRNAs (28S, 18S, 5.8S) • RNA polymerase II (RNA pol II)- synthesizes mRNAs & most small nuclear RNAs (snRNAs & snoRNAs) • RNA polymerase III (RNA pol III) - synthesizes various small RNAs (tRNAs, 5S rRNA & U6 snRNA)

14. Transcription • Eukaryotic Transcription • Much of what we know is derived from studies of RNA pol II from yeast • 1. Seven more subunits than its bacterial RNA pol • 2. The core structure & the basic mechanism of transcription are virtually identical • 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins • 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)

15. Transcription • Eukaryotic Transcription • Much of what we know is derived from studies of RNA pol II from yeast • 1. Seven more subunits than its bacterial RNA pol • 2. The core structure & the basic mechanism of transcription are virtually identical • 3. Additional subunits of eukaryotic polymerases are thought to play roles in the interaction with other proteins • 4. Eukaryotes require a large variety of accessory proteins or transcription factors (TFs)

16. Transcription • Eukaryotic Transcription • All major RNA types (mRNA, tRNA, rRNA) must be processed • The final products are derived from precursor RNA molecules that are considerably longer than the final RNA product • The primary (1°) transcript is is equivalent in length to the full length of the DNA transcribed • The corresponding segment of DNA from which 1° transcript is transcribed is called transcription unit • The1° transcript is short-lived; it is processed into smaller, functional RNAs • Processing requires variety of small RNAs (90 – 300 nucleotides long) & their associated proteins

17. Review from last time • Chapter 11 is about two processes: • Transcription – the process of copying a DNA strand into RNA • Translation – the process of producing an amino acid chain from a transcribed RNA • RNA is similar to DNA but with some minor differences • There are several different types of RNA • Without RNA, there can be no gene expression • The promoter is the site of assembly of the transcription apparatus, be familiar with it • Promoters are particular DNA sequences that are bound by transcription factors • Prokaryotic RNA polymerase complexes consist of five components – sigma specifies the promoter sequence used • Eukaryotic transcription is more complex • More components • Three different RNA polymerases with different jobs • In eukaryotes, RNA transcripts must be processed

18. RNA processing • Ribosomes are the location of protein synthesis • They are combinations of protein and RNA and are made up of two parts (small and large subunits) • Millions exist in any given eukaryotic cell • ~80% of RNA in a cell is rRNA • rDNA, typically exists in hundreds of tandemly repeated copies

19. RNA processing

20. RNA processing • Eukaryotic ribosomes have four distinct rRNAs: • Three rRNAs in the large subunit (28S, 5.8S, 5S in humans); • One in the small (18S in humans) subunit • S value (or Svedberg unit) • 28S = ~5000 nucleotides • 18S = ~2000 nucleotides • 5.8S = ~160 nucleotides • 5S = ~120 nucleotides

21. RNA processing • Eukaryotic ribosomes have four distinct rRNAs: • 28S, 5.8S & 18S rRNAs are produced from a single 1° transcript that is transcribed by RNA pol I • 5S rRNA is synthesized from a separate RNA precursor using RNA pol III

22. RNA processing • The likely rRNA processing pathway • Cleavages 1 and 5 remove the ends of the 1° transcript • Two pathways exist for the remaining processing • End result is the same – • 18S + paired 28S/5.8S • 5S is produced by a second transcription unit

23. RNA processing • snoRNAs – small nucleolar RNA • Vital to rRNA processing • Pair with proteins to make snoRNPs • Consist of relatively long stretches (10-21 nucleotides) that are complementary to parts of rRNA transcript • can form double-stranded hybrids • bind to specific portions of pre-rRNA to form an RNA-RNA duplex & guide an enzyme within the snoRNP to modify a particular pre-rRNA nucleotide • ~200 different snoRNAs exist

24. RNA processing • snoRNAs – small nucleolar RNA • snoRNPs associate with rRNA precursor before it is fully transcribed • Best characterized RNP contains U3 snoRNA and >2 dozen different proteins • Binds to precursor 5' end of transcript & catalyzes removal of transcript 5' end

25. RNA processing • 5S rRNA • Transcribed by RNA pol III • Pol III is unique in that utilizes promoters within the transcription unit

26. RNA processing • Transfer RNAs (tRNA) • Responsible for carrying amino acids to the site of protein synthesis • In humans, ~1300 genes for ~50 tRNAs • Human tRNA genes exist on all chromosomes except 22 and Y and are highly clustered on 1, 6, and 7 • Transcribed by RNA pol III

27. RNA processing • Messenger RNAs (mRNA) • Transcribed by RNA pol II • Remember this? • http://www.as.wvu.edu/~dray/219files/Transcription_588x392.swf • Polymerase II promoters lie to 5' side of each transcription unit • In most cases, between 24 & 32 bases upstream from transcription initiation site is a critical site • Consensus sequence that is either identical or very similar to 5'-TATAAA-3‘, the TATA box • The site of assembly of a preinitiation complex • contains the GTFs & the polymerase • must assemble before transcription can be initiated

28. RNA processing • The preinitiation complex • Step 1 - binding of TATA-binding protein (TBP) • Purified eukaryotic polymerase, cannot recognize a promoter directly & cannot initiate accurate transcription on its own • TBP is part of a much larger protein complex called TFIID • TBP kinks DNA and unwinds ~1/3 turn

29. RNA processing • The preinitiation complex • Step 2 – Binding of ~8 TAFs (TBP- associated factors) to make up the complete TFIID complex • Step 3 – Binding of TFIIA (stabilizes TBP-DNA interaction) and TFIIB (involved in recruiting other TFs and RNA pol II)

30. RNA processing • The preinitiation complex • Step 4 – RNA pol II and TFIIF bind via recruitment by TFIIB • Step 5 – TFIIE and TFIIH bind • TFIIH is the key to activating transcription in most cases • TFIIH is a protein kinase – phosphorylates proteins • TFIIH may also act as a helicase

31. RNA processing • The preinitiation complex • All these general transcription factors and pol II are enough to generate basal transcription • Transcription can be upregulated or downregulated by a huge diversity of other cis and trans acting factors to be discussed in chapter 12.

32. Review from last time • All RNA transcripts must be processed. • 3 of the 4 ribosomal RNAs (rRNAs) are transcribed as a single unit and processed by cleaving individual units out • snoRNAs are critical to the rRNA processing • tRNAs and 5S rRNA are transcribed by RNA pol III • RNA pol III genes are unique in having internal promoters • Be aware of the components making up the preinitiation complex of a RNA pol II gene and their roles in transcription initiation • Review of RNA pol II transcription initiation at: • http://www.as.wvu.edu/~dray/219files/TranscriptionAdvanced.wmv • Review of human genome complexity at: • http://www.dnalc.org/ddnalc/resources/chr11a.html

33. RNA processing • mRNA • Transcription generates messenger RNA • A continuous sequence of nucleotides encoding a polypeptide • Transported to cytoplasm from the nucleus • Attached to ribosomes for translation • Are processed to remove noncoding segments • Are modified to protect from degradation and regulate polypeptide production

34. RNA processing • mRNA • RNA polymerase II assembles a 1° transcript that is complementary to the DNA of the entire transcription unit • 1° transcript contains both coding (specify amino acids) and noncoding sequences • Subject to rapid degradation in its raw state

35. RNA processing • mRNA processing – 5’ cap • 5' methylguanosine cap forms very soon after RNA synthesis begins • 1. The last of the three phosphates is removed by an enzyme • 2. GMP is added in inverted orientation so guanosine 5' end faces 5' end of RNA chain • 3. Guanosine is methylated at position 7 on guanine base while nucleotide on triphosphate bridge internal side is methylated at ribose 2' position (methylguanosine cap)

36. RNA processing • mRNA processing – 5’ cap • Possible/known functions of 5’ cap • May prevent exonuclease digestion of mRNA 5' end, • Aids in transport of mRNA out of nucleus • Important role in initiation of mRNA translation

37. RNA processing • mRNA processing – Polyadenlyation • The poly(A) tail – 3' end of most mRNAs contain a string of adenosine residues (100-250) that forms a tail • Protects the mRNA from degradation • AAUAAA signal ~20 nt upstream from poly(A) addition site • Poly(A) polymerase, poly(A) binding proteins, and several cleavage factors are involved • http://www.as.wvu.edu/~dray/219files/mRNAProcessingAdvanced.wmv

38. RNA processing • mRNA processing – Splicing • Requires break at 5' & 3' intron ends (splice sites) & covalent joining of adjacent exons (ligation) • http://www.as.wvu.edu/~dray/219files/mRNASplicingAdvanced.wmv • Why introns? • Disadvantages – extra DNA, extra energy needed for processing, extra energy needed for replication • Advantages – modular design allows for greater variation and relatively easy introduction of that variation

39. RNA processing • mRNA processing – Splicing • Splicing MUST be absolutely precise • Most common conserved sequence at eukaryotic exon-intron borders in mammalian pre-mRNA is G/GU at 5' intron end (5' splice site) & AG/G at 3' end (3' splice site)

40. RNA processing • mRNA processing – Splicing • Sequences adjacent to introns contain preferred nucleotides that play an important role in splice site recognition

41. RNA processing • mRNA processing – Splicing • Nuclear pre-mRNA (common) • snRNAs + associated proteins = snRNPs • snRNAs – 100-300 bp • U1, U2, U4, U5, U6 • 3 functions for snRNPs • Recognize sites (splice site and branch point site) • Bring these sites together • Catalyze cleavage reactions • Splicosome – the set of 5 snRNPs and other associated proteins • Summary movie available at: • http://www.as.wvu.edu/~dray/219files/mRNAsplicing.swf

42. Review from last time • Messenger RNAs (mRNAs) experience three processing steps • Addition of a methylguanosine cap • Polyadenylation • Splicing • Be familiar with the characteristics and functions of the 5’ cap • Be able to describe the polyadenylation signals on an mRNA, the functions of the proteins involved, and the process of polyadenylation • Be able to describe the nature of the splicosome • Be able to describe the sequence landmarks required for accurate splicing

43. RNA processing • mRNA processing – Splicing • 1. U1 and U2 snRNPs bind via complementary RNA sequences • Note the A bulge produced by U2 • U2 is recruited by proteins associated with an exon splice enhancer (ESE) within the exon

44. RNA processing • mRNA processing – Splicing • 2. U2 recruits U4/U5/U6 trimer • U6 replaces U1, U1 and U4 released • U5 binds to upstream exon

45. RNA processing • mRNA processing – Splicing • 3. U6 catalyzes two important reactions • Cleavage of upstream exon from intron (bound to U5) • Lariat formation with A bulge on intron • Exons are ligated • U2/U5/U6 remain with intron

46. RNA processing • mRNA processing – Splicing • Several lines of evidence suggest that it is the RNA in the snRNP that actually catalyzes the splicing reactions • 1. Pre-mRNAs are spliced by the same pair of chemical reactions that occur as group II (self-splicing) introns • 2. The snRNAs needed for splicing pre-mRNAs closely resemble parts of the group II introns • Proteins likely serve supplemental functions • 1. Maintaining the proper 3D structure of the snRNA • 2. Driving changes in snRNA conformation • 3. Transporting spliced mRNAs to the nuclear envelope • 4. Selecting the splice sites to be used during the processing of a particular pre-mRNA

47. RNA processing • mRNA processing – Splicing • Group II intron self-splicing summary (rare)

48. RNA processing • Implications of RNA catalysis and splicing • The RNA world • Which came first, DNA or protein?... Apparently, it could have been RNA • Information coding AND catalyzing ability • Alternative splicing • Allows one gene to encode multiple protein products • Intron sequences actually encode some snoRNAs • Evolutionary innovation • Exon shuffling

49. RNA processing • Small noncoding RNAs and RNA silencing • To study the effect of disabling a gene, researchers have had to produce ‘knockouts’ through a difficult, time consuming process involving some random chance. • …until the discovery of RNA interference • introduce dsRNA for the gene to be silenced and the mRNAs for that gene are destroyed

50. 10_38_ES.cells.jpg • …until the discovery of RNA interference • introduce dsRNA for the gene to be silenced and the mRNAs for that gene are destroyed