Genes & Chromosomes Part III, Chapters 24, 25

Central Dogma DNA replicates  more DNA for daughters (Gene w/in) DNA transcribed  RNA –Gene = segment of DNA –Encodes info to produce funct’l biol product RNA translated  protein

Genome Sum of all DNA –Genes + noncoding regions Chromosomes –Each w/ single, duplex DNA helix –Contain many genes Historical: One gene = one enzyme Now: One gene = one polypeptide Some genes code for tRNAs, rRNAs Some DNA sequences (“genes”) = recognition sites for beginning/ending repl’n, transcr’n

Most gene products are “proteins” –Made of aa’s in partic sequence –Each aa encoded in DNA as 3 nucleotide seq along 1 strand of dbl helix –How many nucleotides (or bp’s) needed for prot of 350 aa’s?

Prokaryotic DNA Viruses –Rel small amt DNA 5K to 170K base pairs (bp’s) –One chromosome Chromosome = “packaged” DNA –Many circular

Bacterial DNA -- larger than viral –E. coli ~4.6 x 10 6 bp’s –Both chromosomal, extrachromosomal Usually 1 chromosome/cell Extrachromosomal = plasmid – bp’s –Replicate –Impt to antibiotic resistance

Chromosomes Complex Packaging reduces E.coli DNA 850x

Eukaryotic DNA Many chromosomes –Single human cell DNA ~ 2 m Must be efficiently packaged

Euk Chromosomes Prok’s – usually only 1 cy of each gene (but exceptions) Euk’s (ex: human) –Book: coding region (genes coding for prot’s) ~ 1.5% total human genome Exons

Euk’s (ex: mouse): ~30% repetitive –“Junk”? –Non-transcribed seq’s Centromeres – impt during cell division Telomeres – help stabilize DNA Introns – “intervening” seq’s –Function unclear –May be longer than coding seq’s (= exons)

Supercoiling DNA helix is coil –Relaxed coil not bent –BUT can coil upon itself  supercoil Due to packing; constraints; tension Superhelical turn = crossover Impt to repl’n, transcr’n –Helix must relax so can open, expose bp’s –Must unwind from supercoiling

Topoisomerases Enz’s in bacteria, euk’s Cleave phosphodiester bonds in 1/both strands –Where are these impt in nucleic acids? –Type I – cleaves 1 strand –Type II – cleaves both strands After cleavage, rewind DNA + reform phosphodiester bond(s) Result – supercoil removed

Type I

Type II

DNA Packaging Chromosomes = packaged DNA –Common euk “X”- “Y”-looking structures –Each = single, uninterrupted mol DNA Chromatin = chromosomal material –Equiv amts DNA + protein –Some RNA also assoc’d

1 st Level Pakaging in Euk’s Around Histones DNA bound tightly to histones

Basic prot’s About 50% chromosomal mat’l 5 types –All w/ many +-charged aa’s –Differ in size, amt +/- charged aa’s What aa’s are + charged? Why might + charged prot be assoc’d w/ DNA helix? 1 o structures well conserved across species Histones

Must remove 1 helical turn in DNA to wind around histone –Topoisomerases impt

Histones specific locations on DNA –Mostly AT-rich areas

Nucleosome Histone wrapped w/ DNA –  7x compaction of DNA Core = 8 histones (2 copies of 4 diff histone prot’s) ~140 bp DNA wraps around core Linker region -- ~ 60 bp’s extend to next nucleosome Another histone prot may“sit” outside –Stabilizes

Chromatin Further- structured chromosomal mat’l Repeating units of nucleosomes “Beads on a string” –Flexibly jointed chain

30 nm Fiber Further nucleosome packing ~100x compaction Some nucleosomes not inc’d into tight structure

Rosettes Fiber loops around nuclear scaffold –Proteins + topoisomerases incorporated K bp’s per loop –Related genes in loop Book ex: Drosophila loop w/ complete set genes coding for histones ~6 loops per rosette = ~ 450K bp’s/ rosette Further coiling, compaction   10,000X compaction total

Semiconservative Replication 2 DNA strands/helix Nucleotide seq of 1 strand automatically specifies complementary strand seq –Base pairing rule: A w/ T and G w/ C ONLY in healthy helix –Each strand serves as template for partner “Semiconservative” –Semi – partly –Conserved parent strand

DNA repl’n  daughter cell w/ own helix –1 strand is parental (served as template) –2 nd strand is newly synth’d

Definitions Template –DNA strand w/ precise info for synth complementary strand –= parental strand during repl’n Origin –Unique point on DNA helix which repl’n begins Replication Fork –Site of unwinding of parental strand and synth of daughter strand NOTE: helix unwinding crucial to repl’n success

Repl’n Fork – cont’d –Bidirectional repl’n 2 repl’n forks simultaneously synth daughter strands

At Replication Fork Both parental strands serve as templates –Simultaneous synth of daughter cell dbl helices Expected –Helix unwinds  repl’n fork –Get 2 free ends 1 end 5’ –PO 4, 1 end 3’ –PO 4 REMEMBER: paired strands of helix antiparallel

Expected -- cont’d –Repl’n each strand at end of parent One strand will replicate 5’  3’ –Direction of active repl’n 5’  3’ parent strand w/ 3’ end –Yields 2 nd antiparallel dbl helix One strand will replicate 3’  5’ –Direction of active repl’n 3’  5’ parent strand w/ 5’ end –Yields antiparallel dbl helix

But, exper’l evidence: –Repl’n ALWAYS 5’  3’ Can envision at parental strand w/ 3’ end What happens at other parental strand??

Okazaki Fragments Discovered by Dr. Okazaki –Found near repl’n fork Small segments daughter strand DNA synth’d 5’  3’ –Along parental template strand w/ 5’ end Get series small DNA segments/fragments –So synth along this strand in opp direction of overall replication (or of unwinding of repl’n fork)

“Lagging strand” –Takes longer to synth fragments + join them Other parental strand, w/ continuous synth “leading strand” W/ repl’n, fragments joined enzymatically  complete daughter strand Overall, repl’n on both strands in 5’  3’ direction (w/ respect to daughter)

Don’t be confused w/ bi-directional repl’n –Bidirectional: >1 repl’n fork initiating repl’n simultaneously –At each fork, repl’n takes place along both strands –At each fork, repl’n in 5’  3’ direction ONLY along each strand

Enz’s That Degrade DNA Exonucleases – degrade DNA from one end of molecule –Some digest one strand 3’  5’ –Some digest in 5’  3’ direction Endonucleases – degrade DNA from any site