Structure and Organization of Nucleic Acids Flashcards
4 types of non-coding DNA
- Spacer sequences (separating genes)
- Introns
- Genes encoding nonprotein-coding RNAs (rRNA, snRNA, snoRNA, and miRNA)
- Repetitious DNA (most abundant type)
Types of repetitious DNA
- Satellite DNA (simple-sequence DNA) = comprised of identical repeats of short sequences arranged in tandem arrays extending for 20-100 thousand bps, mostly confined to centromere and telomere structures of chromosomes
- Interspersed repeats = most abundant class of repetitious DNA, approximately 45% of human DNA genome and comprises of transposons and retrotransposons
Distinguish between exons and introns.
Exons = sequences that are translated into protein Introns = sequences that are spliced out of the primary RNA transcript and not transcribed into protein
How does a gene family arise? Define it.
Arise from gene duplication events; contain genes with similar nucleotide sequences and encode similar (but not identical) proteins; can be clustered at one chromosomal locus or dispersed through genome. Includes pseudogenes and genes with changed functions as they acquire mutations.
What is meant by the term pseudogene and how does it relate to gene families?
Pseudogenes are made when a duplicated gene in a gene family accumulates mutations that alter the functional characteristics in order to inactivate it. (Key here is that they are INACTIVATED)
Example of a gene family
β-globin gene family: clustered family located on chromosome 11 consisting of 5 genes (2 adult globins β and δ, 2 fetal globins Aɣ and Gɣ, 1 embryonic globin ε, and two pseudogenes ψβ1 and ψβ2
What is chromatin?
Complex of condensed DNA (~1/3) and protein (~2/3)
Describe the three levels of compaction that allow enormously long DNA molecules to be packaged into the nucleus of the cell.
- DNA is wrapped around histone proteins (histone + DNA = nucleosome, ~11 nm) – sealed by fifth histone (H1) attaching
- Nucleosomes form solenoid fiber (30 nm wide)
- 30 nm fiber forms 300 wide loop domains
* *After cell division looped domains attach to protein scaffold to form 700 nm wide fibers**
How many chromosomes are found in a diploid human cell?
46 chromosomes (2 sex chromosomes, 44 autosomes)
Contrast the mitochondrial genome and the nuclear genome.
The mitochondrial genome is…
- -circular (nuclear is straight)
- -only 16.6 kilobases (nuclear has over 6 billion base pairs)
- -present in multiple (5-10) copies per organelle (nuclear has only 2 copies of each chromosome per cell)
- -higher gene density than nuclear genome (encodes 13 proteins and 24 RNA molecules)
- -not extensively associated with proteins
- -more similar to prokaryotic chromosomes
What is meant by the terms “heterochromatin” and “euchromatin”?
Refers to the organization/compartmentalization of chromosomes in the nucleus.
Heterochromatin – stains intensively, indicating that it is complexed with proteins, highly condensed and transcriptionally inactive; concentrated at periphery of nucleus
Euchromatin – appears translucent when stained, indicating that it contains transcriptionally active, decondensed DNA
What processes occur in the nucleolus?
Assembly of ribosomal subunits (in the periphery) – nucleolus contains ~200 copies of 3 rRNA genes on 5 chromosomes, which are actively transcribed for this process
Describe the key features of nuclear pore complex.
- -Nuclear basket (nucleoplasm side)
- -Protein ring anchored in double membrane
- -Central transporter inside protein ring (limits diameter of the opening to regulate)
- -Filaments (cytoplasm side)
Nuclear pore complex
- -Large macromolecular complex in the nuclear envelope
- -Consists of 50-100 proteins
- -Functions to regulate traffic between nucleus and cytoplasm
Describe the Ran-dependent import of material to the nucleus.
- Protein needing to be imported binds to importin
- Importin associates with Ran-GTP inside the nucleus, released imported protein
- Importin/Ran complex is transported out of nucleus
- Ran-bound GTP is hydrolyzed, releasing importin from Ran (Ran-GDP reenters to be converted again)
Describe the Ran-dependent export of material from the nucleus.
- Protein needing to be exported binds to exportin and Ran-GTP
- Ran-GTP/exportin complexes are transported out of nucleus
- Hydrolysis of Ran-bound GTP leads to dissociation of the complex (Ran-GDP is exported back to nucleus)
3 types of molecules that must be actively/selectively transported
- mRNA molecules (in)
- Proteins of the cytoplasm (in)
- Ribosomal subunits (out)
Semi-conservative replication
Each duplicated helix of DNA contains one new, synthesized strand and one old, inherited strand
Replication origin
Specific location on chromosome where replication begins (human DNA has multiple)
Replication fork
Where the DNA splits to be replicated
What are the nucleotide donors for DNA synthesis?
dATP, dGTP, dCTP, dTTP
What is the significance of the pyrophosphate formed during chain elongation?
Released as each nucleotide is incorporated; its hydrolysis drives the reaction to the right
What key features are shared by DNA polymerases?
- -Template-directed
- Require primer (cannot initiate synthesis of DNA chain, only elongate an existing chain by adding to free OH group on 3’ end)
- -Synthesize DNA only in 5’ –> 3’ direction
- -use specific 4 dNTPs as substrates
Outline the steps involved in DNA replication.
- Origin recognition complexes (ORC) mark origins of replication, then inactivated
- DNA helicases unwind DNA double helix
- Single-stranded binding proteins bind to single strands of DNA
- Topoisomerases relieve supercoiling
- DNA pol alpha synthesizes short RNA primer
- Polymerase switching occurs
- Accessory proteins are loaded onto DNA molecule next to primer
- 3’-OH end is synthesized continuously (leading strand)
- Primers are synthesized by primase activity and elongated to generate Okazaki fragments
- RNA primers are removed and DNA pol fills gaps
- Looping process allows polymerases to head the same way
What is the role of helicase?
ATP-dependent enzymes that use free energy of ATP hydrolysis to power unwinding of DNA double helix
Why are the single-stranded DNA binding proteins important?
Prevent re-annealing of the DNA strands
What are topoisomerases and what do they do?
Proteins that relieve supercoiling and allow replication to progress
Outline the reactions catalyzed by topoisomerase I that relieve supercoiling.
Cuts phosphodiester backbone of one strand, allows other strand to rotate, then re-ligates phosphodiester backbone
What is the significance of the RNA primer?
Required for chain elongation
How are both strands of DNA replicated at the same time and in the same overall direction?
Leading strand: 3’-OH of primer will point TOWARDS the growing replication fork and is synthesized continuously by DNA polymerase ε
Lagging strand: 3’-OH of primer points AWAY from replication fork, so strand is synthesized in opposite direction by DNA polymerase δ and as new stretches of DNA are exposed, new primers are synthesized to continue (generates Okazaki fragments)
Leading strand
Synthesized in the direction of the growing replication fork
Lagging strand
Synthesized in the opposite direction of replication fork using Okazaki fragments
Okazaki fragments
DNA segments that are made for the lagging strand; require multiple RNA primers, which will then requires removal, and ligase activity to join the strands together
How are primers removed and Okazaki fragments joined together?
Specific ribonucleases (enzymes that degrade RNA) remove RNA primers and DNA polymerase fills in gaps; fragments are joined by DNA ligase (requires ATP)
Telomeres
Ends of chromosomes; in humans, DNA sequence is repeated copies of TTAGGG (~10,000 nucleotides)
How are telomeres replicated?
Telomerase (enzyme) binds, using its RNA template molecule to extend the 3’-end of the molecule; process is completed by DNA polymerase on lagging strand
Describe how 3’ and 5’ ends of DNA are defined.
Phosphates are bound to the 3’ and 5’ carbons of the sugar in nucleic acids, forming a diester bond; ends are defined by the carbon group that is unbound to another nucleic acid
Which sugar is found in DNA?
Deoxyribose (2’ carbon)
Which sugar is found in RNA?
Ribose (2’ carbon)
How do you calculate the amounts of all 4 nucleotide bases in DNA when given only the amount of one?
Adenine and thymine are always paired, as are cytosine and guanine. Therefore, if you know the amount of A is 20%, the amount of T will be 20%, and guanine and cytosine will be 30%.
Explain how structure of major groove of DNA allows proteins to bind DNA in sequence-specific manner.
Portions of the bases are exposed in the grooves, which are thus lined with potential for hydrogen bond donor and acceptor sites, allowing proteins to recognize and bind specific DNA sequences (this is where most protein-DNA interactions take place).
DNA intercalating agents
Class of mutagenic molecules that fit exactly on the rungs of the ladder of DNA, distorting the double-helix; most often cause insertions and deletions. Ex. Acridine dyes (acridine orange), ethidium bromide, doxorubicin.
DNA supercoiling
Adding or subtracting twists on the DNA done by some DNA; can be negative (twisted in direction that unwinds helix) or positive (twisted in the direction that winds DNA even more, which occurs during DNA replication or transcription because of unwinding at helix site).
Topoisomerase I
Cuts phosphodiester bond of one strand of DNA in double helix, allowing other to rotate, then re-ligates backbone to seal strand break
Topoisomerase II
Causes double strand break in double helix by cutting both phosphodiester backbones, then re-ligates after other double helix has passed through
Doxorubicin
Acts on human topoisomerase II; used as anticancer agent
Nalidixic acid
Acts on bacterial topoisomerase II and functions as antibiotic
Ciproflaxacin
Acts on bacterial topoisomerase II and functions as antibiotic
How do topoisomerase inhibitors work?
Inhibit ligase activity of type II topoisomerases, leading to accumulation of DNA double strand breaks, which will likely cause cell death (ex. doxorubicin, nalidixic acid, ciproflaxacin)
