CHAPTER 24 Genes and Chromosomes

1. CHAPTER 24Genes and Chromosomes • Organization of information in chromosomes • DNA supercoiling • Structure of the chromosome Key topics:

2. Management and Expression of Genetic Information • Previous chapters dealt with • metabolic pathways, in which the chemical structures of small molecules were modified by enzymes • signal transduction pathways, in which interactions of ligands with receptor proteins caused physiological responses • The following chapters deal with • information pathways, in which genetic information stored as the nucleotide sequence is maintained and expressed

3. Molecular Biology vs. Biochemistry • Biochemistry began with metabolism. First small molecules, then enzymes (proteins) • The discovery of double-helical structure of DNA in 1953 laid a foundation to thinking of biomolecules as carriers of information • It was well understood by 1950 that proteins play roles of catalysts but their role in information transfer was unclear • Francis Crick proposed in 1956 that “Once information has got into a protein it can’t get out again” • The Central Dogma was proposed by Francis Crick at the time when there was little evidence to support it, hence the “dogma”

4. DNA => RNA => Protein The Central Dogma • Information from parental DNA is copied to daughter DNA with high fidelity via DNA replication • RNA is synthesized using DNA as a template during transcription • Viruses are able to make RNA and DNA using RNA as a template in reverse trascription • Proteins are synthesized based on the information stored in ribonucleotide triplets in RNA

5. Molecular Templates for Information Transfer • In transcription, one strand of double stranded DNA acts as molecular template for RNA synthesis • In translation, the triplets of nucleotides in mRNA are matched with corresponding amino acids via triplets of tRNA • Protein sequence determines its biological function

6. Codon groupings of similar side chains GG[GACU] code for Glycine UGG codes for Tryptophan UGA, UAG, UAA are stop codons AG[CU] and UC[GACU] code for Serine The Genetic Code

7. Genomic DNA – extremely long, Packaged for protection • The linear dimensions of DNA are much bigger than the virions or cells that contain them • Bacteriophage T4 viral particle - 0.2 m x 0.1 m • Fully extended T4 DNA double helix (200kbp) ~60 m • DNAin vivo is organized into compact forms, typically via coiling and association with proteins • Naked genomic DNA is very fragile – No intact viral DNA genomes at first • Still nearly impossible to study intact protein-free chromosomes

9. Chromosomes • A Chromosome consists of one covalently connected DNA molecule and associated proteins • Viral genomic DNA may be associated with capsid proteins • Prokaryotic DNA (circular) is associated with proteins in the nucleoid • Eukaryotic chromosomes (linear) organized with proteins into chromatin

10. Bacterial Genome Usually Consists of One Circular Chromosome • Extra chromosomal elements • Mobile elements • Plasmids smaller circular DNA molecules • Copy numbers 1-hundreds • Temperate phages • Plasmid replicons • Integrated • Mobile elements • Insertion sequences • Transposons

11. Karyotype visible after condensation, during mitosis Some Ciliates have macro nuclei and gene sized mini chromosomes Eukaryotic DNA – Multiple Linear Chromosomes

12. Mitochondria and Chloroplasts Have Their Own DNA Circular (Recall the endosymbiont hypothesis)

13. Genome Size and Number of Chromosomes Varies in Species • Estimates from 2008 place the number of human genes to 20,500

14. DNA, Chromosomes, Genes, and Complexity • Neither total DNA, nor number of chromosomes correlates strongly with complexity • Amphibians have much more DNA than humans • Plants have more genes than humans • The correlation between complexity and genome size is poor because most of eukaryotic DNA is non-coding • Recent genome engineering suggests that a minimal living organisms could get by with less than 400 genes

15. Eukaryotic Genes are processed before translation (Introns) • Introns not present in mature RNA – reading frames not maintained • Exon sequences are spliced => mature mRNA • Regulatory sequences direct splicing machinery to the E/I boundaries • Nearly all eukaryotic genes contain introns

16. Some Bacterial Genomes Also Contain Introns • It was thought until 1993 that introns are exclusive feature of eukaryotic genes • About 25% of sequenced bacterial genomes show presence of introns • Introns in bacterial chromosome do not interrupt protein-coding sequences; they interrupt mainly tRNA sequences • Introns in phage genomes within bacteria interrupt protein-coding sequences • Many bacterial introns encode for catalytic RNA molecules that have ability to insert and reverse transcribe themselves into the genomic DNA • Splicing mechanisms conserved

17. Composition of the Human Genome • Only a small fraction (1.5 %) of the total genome codes for proteins • Non protein-coding sequences • Some regulate gene expression • Some encode RNA • Stable and regulatory • Historical junk • (mobile elements, gene fragments, evolutionary building blocks?)

18. Important Structural Elements of the Eukaryotic Chromosome • Telomeres compensate for incomplete replication of the ends of linear, distinguish ends from breaks • Centromere (one per chromosome) guide chromosome partitioning to daughter cells • Origins allow for replication initiation

19. Telomeres, Telomerase, Aging, Cancer, and Immortality • Telomere sequences identified in protozoan mini-chromosomes • Telomerase solves the end-replication problem • In tissues lacking telomerase, telomeres are shortened after each round of replication • Germ-line cells, stem cells, cancer cells, immortalized cell lines express • Human somatic cells divide until telomere sequences are lost, losing ability to divide again (Hayflick limit)

20. DNA Supercoiling • Transitions from DS to SS DNA change Twist • Local Twist can be propagated to DNA ends in short linear fragments • But not circular or long anchored molecules

21. Supercoiling of Plasmid DNA

22. Linking Number • Linking number in relaxed DNA: number of base pairs divided by the number of base pairs per helical turn • Relaxed circular dsDNA of 2,100 base pairs in the B form (10.5 base pairs per turn) has linking number of 200

23. Superhelical Density σ σ = Δ Lk / Lk0 Typically negative -0.05 to -0.07

24. Link = Twist + Writhe

25. Topoisomers • Compact negatively supercoiled DNA travels fastest in the agarose gel electrophoresis experiment

26. Topoisomerases • Enzymes that change linking number of DNA are called topoisomerases • These enzymes are required for DNA unwinding and rewinding during transcription and replication • There are two major types of topoisomerases • Type I topoisomerases work by making a transient cut in one DNA strand • Type II topoisomerases work by making a transient cut in both DNA strands

27. Type I Topoisomerases • Type I topoisomerases: • Sub Tyr-Phospho diester for ribose • pass the intact strand through the gap • Rejoin Phospho diester backbone • Delta Lk = 1

28. Topoisomerase II (Gyrase) • Type II topoisomerases • break both strands • pass a DNA segment through the break • join the strands • change linking number in increments of • “Etherializes” DNA • N. Cozzarelli

29. Topoisomerases are Targets for Antibiotics and Anti-cancer Drugs • All cells need topoisomerase • Hyper-active replication makes cancer cells more sensitive • DNA breaks

30. Solenoidal supercoils resemble nucleosome wrapping

31. Cell Cycle Chromatin condensation for mitosis Chromatin expansion for replication and transcription

32. Beads on a string (expanded)

34. Histone Octamer

35. Nucleosome Core Structure

36. Global and local effects

37. Histone Variants

38. Chromatin Immunoprecipitation

42. Chapter 24: Summary In this chapter, we learned that: • Genetic information of the cell is encoded in the nucleotide sequence of one or several DNA molecules • The protein-coding regions represent only a small fraction of the total DNA • Telomeres and centromeres regulate cell division • Bacterial DNA is usually supercoiled for efficient packing • Eukaryotic DNA is wound around positively charged histones • Higher order organization of chromatin likely involves coils upon coils upon coils …