L37: Organization of the Genome

Question 1

What is non-coding DNA? List 4 types

Answer 1

• Regions of DNA that do not encode for proteins 1.) Introns 2.) Genes encoding RNAs 3.) Satellite DNA (found at centromere and telomere) 4.) Interspersed repeats (transposons and retrotransposons)

Question 2

Distinguish bw exons and introns

Answer 2

• Exon: sequence of gene translated into protein - Intron: sequence of gene removed from primary RNA

Question 3

What is meant by the terms gene family and pseudogenes? Give an example of a gene family. How might gene families arise?

Answer 3

• Gene family: genes with similar nt sequences encoding similar but not identical proteins, arise through gene duplications events - Pseudogenes: non-functional copies of genes that cannot encode functional protein - Example: beta-globin on c/s 11

Question 4

What is chromatin? Describe the three levels of compaction that allow enormously long DNA molecules to be packages into the nucleus of the cell

Answer 4

• Chromatin is DNA associate with proteins. This allows for large genome to be condensed and to fit inside cell nucleus - 1.) DNA wraps around histone proteins to form nucleosome - 2.) Collections of nucleosomes form 11 nm fiber - 3.) Nucleosomes associate further to form 30 nm fiber - 4.) 30 nm fiber forms 300 nm wide loop domains - 5.) Loop domains associate with protein scaffold 700 nm fiber forms

Question 5

Describe mitochondrial genome

Answer 5

• Mitochondrial: small, circular, 5-10 copies per mitochondrion, encodes resp chain proteins, tRNA and rRNA molecules, no histones

Question 6

What is mean by the terms heterochromatin and euchromatin?

Answer 6

• Heterochromatin: DNA extensively associated with protein and highly condensed, transcriptionally inactive - Euchromatin: decondensed and transcriptionally active

Question 7

What processes occur in the nucleolus?

Answer 7

• Nucleolus are clusters of genes encoding rRNAs - This is site of ribosome assembly

Question 8

Describe key features of the nuclear pore complex

Answer 8

• Cytoplasmic filaments - Nuclear basket - Cytoplasmic ring - Nuclear ring - Central transporter

Question 9

How is material transported into and out of the nucleus?

Answer 9

• Small molecules diffuse through aqueous channel formed by nuclear pore complexes - Proteins, ribosomal subunits, tRNAs, some mRNA transport is tightly regulated - Import mediated by G-protein Ran and importin - Export mediated by G-protein Ran and exportin

Question 10

Is mRNA export under Ran regulation?

Answer 10

• most mRNAs are independent of Ran

Question 11

Explain transportation from cytoplasm to nucleus

Answer 11

• Ran-GTP bound to importin = inactivated Importin - Ran hydrolyzes GTP to GDP and importin dissociates, Ran-GDP moves into nucleus - Importin binds cargo and moves through nuclear pore complexes into nucleus - Ran-GDP in nucleus binds GTP and dislocates GDP - Importin dissociates from cargo in nucleus and associates with Ran-GTP moves out to cytoplasm

Question 12

Explain transportation from nucleus to cytoplasm

Answer 12

• Ran-GDP binds GTP and dissociates from GDP forming Ran-GTP - Ran-GTP binds exportin, binds cargo and moves through nuclear pore complex into cytoplasm - Hydrolysis of complex occurs, Cargo, exportin and Ran-GDP dissociate, Ran-GDP returns into nucleus

Question 13

What key features are share by DNA polymerases?

Answer 13

• Template-directed - Require a primer containing free 3’OH - Synthesize DNA only in 5-3’ direction - Use dATP, dGTP, dCTP and dTTP as substrates

Question 14

Outline the steps involved in replication of DNA

Answer 14

1. Origin recognition complex proteins bind to origins – ORC activated s phase, then inactivated until after mitosis 2. DNA helicase unwind the double helix 3. SSBPs bind DNA and prevent reformation of double helix 4. Tension in DNA acted on by topoisomerase I, which cuts phosphodiester bond of one strand and allows strand to rotate, religation occurs 5. Primase activity of DNA pol alpha uses exposes SS DNA region as template to synthesize a short complimentary stretch of RNA, provides 3’ OH 6. DNA pol alpha is replaced by delta or epsilon 7. Protein complex known as sliding clamp is loaded onto DNA, facilitates pol activity 8. DNA pol replicates continuously on leading strand, discontinuously (delta) on lagging strand 9. RNase activity removes primer, DNA pol possibly delta fills in gap left by primer removal 10. DNA ligase joins Okazaki fragments

Question 15

What is the role of DNA helicase?

Answer 15

• Starts at origins and begins unwinding the double helix

Question 16

Why are single stranded DNA binding proteins important?

Answer 16

• bind DNA and prevent reformation of the double helix

Question 17

What are topoisomerases and what do they do? Outline reactions catalyzed by topoisomerase I that relieve supercoiling

Answer 17

• Enzymes that relieve supercoiled loops/tension on DNA by cutting phosphodiester backbone - Topo I has OH group that cleaves phosphodiester backbone of one strand, becomes bound to strand, allows rotation of other strand and relegates the phosphodiester backbone

Question 18

What is the significance of the RNA primer?

Answer 18

• DNA synthesis cannot occur with- out template - RNA primer is laid down by DNA pol alpha and provides free 3’ OH

Question 19

Given that the two strands of the DNA double helix are antiparallel, and that DNA polymerases only polymerize in the 5’ to 3’ direction, how are both strands replicated at the same time and in the same overall direction?

Answer 19

• DNA loops out and allows two polymerases to move along the template in the same direction, replicating both leading and lagging strands at same time

Question 20

Define leading and lagging strands

Answer 20

• Leading strand: strand of DNA with free 3’ OH pointing towards replication fork - Lagging strand: strand of DNA with free 3’ OH pointing away from replication fork

Question 21

What is meant by Okazaki fragments?

Answer 21

• As a result of discontinuous synthesis of lagging strand as replication fork grows, series of synthesized DNA series exists on this strand

Question 22

How are primers removed and Okazaki fragments joined together?

Answer 22

• Specific RNase activity removes primer - DNA pol (possibly delta) fills in gap left by primer removal - DNA ligase uses ATP to seal Okazaki fragments

Question 23

What are telomeres and how are they replicated?

Answer 23

• Telomeres are ends of linear chromosomes and contain many repeats of TTAGGG - Replicated by enzyme called telomerase, which is a riboprotein, containing RNA molecule that is complementary to telomere sequence Steps: 1.) Telomerase binds to overhanging telomere strand (3’) 2.) Extends 3’ end using RNA template 3.) DNA Pol completes lagging strand synthesis (5’ end)

Question 24

Describe how the 3’ and 5’ ends of a DNA strand are defined

Answer 24

• 3’ end = leading strand = continuous synthesis of DNA - 5’ end = lagging strand = discontinuous synthesis of DNA

Question 25

Which sugar is found in DNA and RNA? To which carbon of the sugar is the base attached?

Answer 25

• DNA has deoxyribose - RNA has ribose - Bases are attached to 1’ position

Question 26

DNA contains 20% of adenine, how much T, C, G is present?

Answer 26

• 20% thymine - 30% guanine - 30% cytosine

Question 27

Explain how the structure of the major groove of DNA allows proteins to bind DNA in a sequence-specific manner.

Answer 27

• Major grooves are result of particular way in which bonds between sugar and base in each base pair are aligned and therefore exposed/seen when looking into molecule - These grooves allow proteins to recognize and bind to specific DNA sequences

Question 28

What are DNA intercalating agents? Name three

Answer 28

• Intercalating agents are molecules that fit into the ladder-like structure of the double-helix causing distortion of it - Acridine orange, ethidium bromide, doxorubicin

Question 29

What is meant by the term supercoiling in relation to DNA?

Answer 29

• DNA assumes a double helix based on interactions between bases - Some enzymes put strain on double-helix by adding or subtracting twists, which results in formation of supercoiled DNA

Question 30

Compare and contrast the mechanism of action of type I and type II topoisomerases

Answer 30

• Topo I: cuts phosphodiester backbone of one strand of DNA in dbl helix, allows rotation, re-ligates - Topo II: cuts phosphodiester backbone of both strands,allows rotation and re-ligates

Question 31

What are the targets of nalidixic acid, cirprofloxacin and doxorubicin?

Answer 31

• Nalidixic acid and ciprofloxacin act on bacterial topoisomerase II enzymes and function as antibiotics - Doxorubicin acts on human topoisomerase II and is used as an anticancer agent