Genome Organization & Protein Synthesis and Processing in Plants

1. Genome Organization & Protein Synthesis and Processing in Plants

2. Viral genomes • Viral genomes: ssRNA, dsRNA, ssDNA, dsDNA, linear or ciruclar • Viruses with RNA genomes: • Almost all plant viruses and some bacterial and animal viruses • Genomes are rather small (a few thousand nucleotides) • Viruses with DNA genomes (e.g.lambda = 48,502 bp): • Often a circular genome. • Replicative form of viral genomes • all ssRNA viruses produce dsRNA molecules • many linear DNA molecules become circular • Molecular weight and contour length: • duplex length per nucleotide = 3.4 Å • Mol. Weight per base pair = ~ 660

3. Procaryotic genomes • Generally 1 circular chromosome (dsDNA) • Usually without introns • Relatively high gene density (~2500 genes per mm of E. coli DNA) • Contour length of E.coli genome: 1.7 mm • Often indigenous plasmids are present

4. -lactamase Plasmids ori Extra chromosomal circular DNAs • Found in bacteria, yeast and other fungi • Size varies form ~ 3,000 bp to 100,000 bp. • Replicate autonomously (origin of replication) • May contain resistance genes • May be transferred from one bacterium to another • May be transferred across kingdoms • Multicopy plasmids (~ up to 400 plasmids/per cell) • Low copy plasmids (1 –2 copies per cell) • Plasmids may be incompatible with each other • Are used as vectors that could carry a foreign gene of interest (e.g. insulin) foreign gene

5. Eukaryotic genome • Moderately repetitive • Functional (protein coding, tRNA coding) • Unknown function • SINEs (short interspersed elements) • 200-300 bp • 100,000 copies • LINEs (long interspersed elements) • 1-5 kb • 10-10,000 copies

6. Eukaryotic genome • Highly repetitive • Minisatellites • Repeats of 14-500 bp • 1-5 kb long • Scattered throughout genome • Microsatellites • Repeats up to 13 bp • 100s of kb long, 106 copies • Around centromere • Telomeres • Short repeats (6 bp) • 250-1,000 at ends of chromosomes

7. Eucaryotic genomes • Located on several chromosomes • Relatively low gene density (50 genes per mm of DNA in humans) • Contour length of DNA from a single human cell = 2 meters • Approximately 1011 cells = total length 2 x 1011 km • Distance between sun and earth (1.5 x 108 km) • Human chromosomes vary in length over a 25 fold range • Carry organelles genome as well

8. Mitochondrial genome (mtDNA) • Multiple identical circular chromosomes • Size ~15 Kb in animals • Size ~ 200 kb to 2,500 kb in plants • Over 95% of mitochondrial proteins are encoded in the nuclear genome. • Often A+T rich genomes. • Mt DNA is replicated before or during mitosis

9. Chloroplast genome (cpDNA) • Multiple circular molecules • Size ranges from 120 kb to 160 kb • Similar to mtDNA • Many chloroplast proteins are encoded in the nucleus (separate signal sequence)

10. “Cellular” Genomes Viruses Procaryotes Eucaryotes Nucleus Capsid Plasmids Viral genome Bacterial chromosome Chromosomes (Nuclear genome) Mitochondrial genome Chloroplast genome Genome: all of an organism’s genes plus intergenicDNA Intergenic DNA = DNA between genes

11. Estimated genome sizes mammals plants fungi bacteria (>100) mitochondria (~ 100) viruses (1024) 1e1 1e2 1e3 1e4 1e5 1e6 1e7 1e8 1e9 1e10 1e11 1e12 Size in nucleotides. Number in ( ) = completely sequenced genomes

12. Size of genomes

13. Chromosome organization Eucaryotic chromosome Centromere Telomere Telomere p-arm q-arm • Centromere: • DNA sequence that serve as an attachment for protein during mitosis. • In yeast these sequences (~ 130 nts) are very A+T rich. • In higher eucaryotes centromers are much longer and contain • “satellite DNA” • Telomeres: • At the end of chromosomes; help stabilize the chromosome • In yeast telomeres are ~ 100 bp long (imperfect repeats) • Repeats are added by a specific telomerase 5’ – (TxGy)n 3’ – (AxCy)n x and y = 1 - 4 n = 20 to 100; (1500 in mammals)

14. Gene classification intergenic region non-coding genes coding genes Chromosome (simplified) Messenger RNA Structural RNA Proteins transfer RNA ribosomal RNA other RNA Structural proteins Enzymes

15. What is a gene ? • Definitions • Classical definition: Portion of a DNA that determines a single character (phenotype) • One gene – one enzyme (Beadle & Tatum 1940): “Every gene encodes the information for one enzyme” • One gene – one protein: “One gene contains information for one protein (structural proteins included) one gene – one polypeptide • Current definition: A piece of DNA (or in some cases RNA) that contains the primary sequence to produce a functional biological gene product (RNA, protein).

16. Coding region Nucleotides (open reading frame) encoding the amino acid sequence of a protein The molecular definition of gene includes more than just the coding region

17. Noncoding regions • Regulatory regions • RNA polymerase binding site • Transcription factor binding sites • Introns • Polyadenylation [poly(A)] sites

18. Gene Molecular definition: Entire nucleic acid sequence necessary for the synthesis of a functional polypeptide (protein chain) or functional RNA

19. Anatomy of a gene • ORF. From start (ATG) to stop (TGA, TAA, TAG) • Upstream region with binding site. (e.g. TATA box). • Poly-a ‘tail’ • Splices. Bounded by AG and GT splice signals.

20. Bacterial genes • Most do not have introns • Many are organized in operons: contiguous genes, transcribed as a single polycistronic mRNA, that encode proteins with related functions Polycistronic mRNA encodes several proteins

21. Bacterial operon What would be the effect of a mutation in the control region (a) compared to a mutation in a structural gene (b)?

22. Eucaryotic genes Hemoglobin beta subunit gene Exon 1 90 bp Intron A 131 bp Exon 2 222 bp Intron B 851 bp Exon 3 126 bp Splicing Introns: intervening sequences within a gene that are not translated into a protein sequence. Collagen has 50 introns. Exons: sequences within a gene that encode protein sequences Splicing: Removal of introns from the mRNA molecule.

23. Regulatory mechanisms • ‘organize expression of genes’ (function calls) • Promoter region (binding site), usually near coding region • Binding can block (inhibit) expression • Computational challenges • Identify binding sites • Correlate sequence to expression

24. Eukaryotic genes • Most have introns • Produce monocistronic mRNA: only one encoded protein • Large

25. Alternative splicing • Splicing is the removal of introns • mRNA from some genes can be spliced into two or more different mRNAs

26. “Nonfunctional” DNA • Higher eukaryotes have a lot of noncoding DNA • Some has no known structural or regulatory function (no genes) 80 kb

27. Types of eukaryotic DNA

28. Duplicated genes • Encode closely related (homologous) proteins • Clustered together in genome • Formed by duplication of an ancestral gene followed by mutation Five functional genes and two pseudogenes

29. Pseudogenes • Nonfunctional copies of genes • Formed by duplication of ancestral gene, or reverse transcription (and integration) • Not expressed due to mutations that produce a stop codon (nonsense or frameshift) or prevent mRNA processing, or due to lack of regulatory sequences

30. Repetitive DNA • Moderately repeated DNA • Tandemly repeated rRNA, tRNA and histone genes (gene products needed in high amounts) • Large duplicated gene families • Mobile DNA • Simple-sequence DNA • Tandemly repeated short sequences • Found in centromeres and telomeres (and others) • Used in DNA fingerprinting to identify individuals

31. Types of DNA repeats Perfect repeats vs degenerate repeats Tandem repeats (e.g. satellite DNA) 5’-CATGTGCTGAAGGCTATGTGCTGCGACG- 3’ 3’-GTACACGACTTCCGATACACGACGCTGC- 5’ Inverted repeats (e.g. in transposons) Loop 5’-CATGTGCTGAAGGCTCAGCACATCGACG- 3’ 3’-GTACACGACTTCCGAGTCGTGTAGCTGC- 5’ Stem • Form stem-loop structures • Palindroms = adjacent inverted repeats • (e.g. restriction sites) • Form hairpin structures Hairpin

32. Repetitive sequences Satellite DNA Chromosomal DNA Caesium chloride density gradient Repeats in the mouse genome

33. DNA repeats and forensics AluSTXa • Gender determination • Standard technique: PCR amplification of the amelogenin locus • (Males = XY => 103 + 109 bp) • AluSTXa Alu insertion on X • AluSTYa Alu insertion on Y M F Suspect 878 bp 556 bp AluSTYa X-Y homologous regions AluSTYa M F Suspect X 528 bp 199 bp Y Alu sequence

34. Mobile DNA • Move within genomes • Most of moderately repeated DNA sequences found throughout higher eukaryotic genomes • L1 LINE is ~5% of human DNA (~50,000 copies) • Alu is ~5% of human DNA (>500,000 copies) • Some encode enzymes that catalyze movement

35. Transposition • Movement of mobile DNA • Involves copying of mobile DNA element and insertion into new site in genome

36. Why? • Molecular parasite: “selfish DNA” • Probably have significant effect on evolution by facilitating gene duplication, which provides the fuel for evolution, and exon shuffling

37. RNA or DNA intermediate • Transposon moves using DNA intermediate • Retrotransposon moves using RNA intermediate

38. Types of mobile DNA elements

39. LTR (long terminal repeat) • Flank viral retrotransposons and retroviruses • Contain regulatory sequences Transcription start site and poly (A) site

41. LINES and SINES • Non-viral retro-transposons • RNA intermediate • Lack LTR • LINES (long interspersed elements) • ~6000 to 7000 base pairs • L1 LINE (~5% of human DNA) • Encode enzymes that catalyze movement • SINES (short interspersed elements) • ~300 base pairs • Alu (~5% of human DNA)

42. Proteins • Most protein sequences (today) are inferred • What’s wrong with this? • Proteins (and nucleic acids) are modified • ‘mature’ Rna • Computational challenges • Identify (possible) aspects of molecular life cycle • Identify protein-protein and protein-nucleic acid interactions

43. Genetic variation • Variable number tandem repeats (minisatellites). 10-100 bp. Forensic applications. • Short tandem repeat polymorphisms (microsatellites). 2-5 bp, 10-30 consecutive copies. • Single nucleotide polymorphisms

44. Single nucleotide polymorphisms • 1/2000 bp. • Types • Silent • Truncating • Shifting • Significance: much of individual variation. • Challenge: correlation to disease

45. Yeast genome • 4.6 x 106 bp. One chromosome. Published 1997. • 4,285 protein-coding genes • 122 structural RNA genes • Repeats. Regulatory elements. Transposons. • Lateral transfers.

46. Yeast protein functions