Intro Biochem 527 Exam 3

Nucleotides and Nucleic Acids, Genes and Chromosomes, and DNA Metabolism

nucleic acids

polymers of nucleotides used for
- storage of genetic info (DNA)
- transmission of genetic info (mRNA)
- processing of genetic info (ribozymes)
- protein synthesis (tRNA and rRNA)

nucleotide function

nucleotide

nucleotide

consist of 3 parts:
- nitrogenous base
- pentose
- phosphate

Number clockwise, starting from N at bottom of ring

purine

nitrogenous base with a 2-ring structure, where one is 6-membered and the other is 5-membered rings
- adenine
- guanine

Number counter-clockwise, starting from N to left of top C in 6-memb ring, then 5-memb ring counts clockwise

pyrimidine

nitrogenous base with 1-ring structure, with a 6-membered ring
- Cytosine
- Thymine (DNA)
- Uracil (RNA)

nucleoside

consist of 2 parts:
- nitrogenous base
- pentose
- NO PHOSPHATE

phosphate group

- negatively charged at neutral pH
- typically attached to 5' position

- nucleic acids are built using the 5'-triphosphate version of the nucleotide

- 2/3 phosphates used for building nucleic acids form a leaving group, and completed nucleic acids contain 1 phosphate

nitrogenous bases

- derivatives of pyrimidine or purine
- nitrogen-containing heteroaromatic molecules
- planar or almost planar structures
- absorb UV light around 250-270 nm
- neutral molecules at pH 7
- all are good H-bond donors and acceptors

- cytosine, guanine, and adenine found within DNA and RNA
- Thymine found only in DNA
- Uracil found only in RNA

adenine

Purine nitrogenous base that pairs with Thymine in DNA

guanine

Purine nitrogenous base that pairs with Cytosine in DNA

cytosine

Pyrimidine nitrogenous base that pairs with Guanine with DNA

thymine

Pyrimidine nitrogenous base that hydrogen bonds with the nucleotide adenine in DNA.

uracil

Pyrimidine Nitrogen base that pairs with adenine in RNA.

B-N-glycosidic bond

Bond formed in nucleotides between the pentose ring and nitrogenous base at anomeric carbon of sugar in B configuration
- quite stable toward hydrolysis, especially in pyrimidines

Bond is formed:
- to position N1 in pyrimidines
- to position N9 in purines

N1

What position is the B-N-Glycosidic bond formed at in Pyrimidines?

N9

What position is the B-N-Glycosidic bond formed at in Purines?

UV absorption of nucleobases

-absorption of UV light at 250-270 nm is due to pi --> pi* electronic transitions

-excited states of common nucleobases decay rapidly via radiationless transitions
---effective photoprotection of genetic material
---no fluorescence from nucleic acids

polynucleotides

A polymer consisting of many nucleotide monomers in a chain; nucleotides can be those of DNA or RNA.
- covalent bonds formed via phosphodiester linkages
- DNA backbone is fairly stable
- RNA backbone is unstable
- Linear polymers
- Directionality

Phosphodiester linkages

covalent bonds that join adjacent nucleotides between the -OH group of the 3' carbon of one nucleotide and the phosphate on the 5' carbon of the next

T

T/F: DNA backbone is fairly stable due to the absence of an -OH group on position 2' of the pentose in the nitrogenous base

2'

RNA backbone is unstable due to the presence of an -OH group on the ______ position of the pentose in the nitrogenous base

directionality

5' end is different from the 3' end
- we read DNA and RNA sequences from 5' -> 3' ends

base pairing

principle that bonds in DNA can form only between adenine and thymine and between guanine and cytosine

(A+G = T+C)

- purines pair with pyrimidines

2

There are ____ H-bonds between Adenine (A) and Thymine (T)

3

There are _______ H-bonds between Guanine (G) and Cytosine (C)

Hydrogen bonding

What holds 2 strands of DNA together?

3400 A

What is the length of 1000 bp of DNA?

3.4 A

Space between each base pair in DNA

20 A

What is the diameter of DNA?

Amstrong (A)

1 x 10^-10 m

A-form DNA

- Shorter than B-form DNA
- RIGHT handed

B-form DNA

- antiparallel
- Watson-Crick bp DNA structure
- RIGHT handed

Z-form DNA

- Skinnier than B-form DNA
- LEFT handed

complementary strands

When 2 strands of DNA will base pair together to form double-stranded helical structure running antiparallel

replication of genetic code

- strand separation occurs first
- each strand serves as a template for the synthesis of a new strand
- a newly made DNA molecule has one daughter strand and one parent strand

ribozyme

ribosome functioning as an enzyme

DNA denaturation

Covalent bonds remain intact
- genetic code remains intact

Hydrogen bonds are broken
- two strands separate

Base stacking is lost
- UV absorbance increases bc base's function of diminishing UV absorbance when in ds form is lost

Can be induced by high temps or change in pH

May be reversible = Annealing

Increases

UV absorbance _______ when bast stacking is lost in DNA

increased

If a melting curve is higher than the other for a single sample of DNA, this means that there is an _________ amount of C-G pairings compared to A-T pairings

factors affecting DNA denaturation

midpoint of melting (TM) depends on base composition
- High CG increases TM

TM depends on DNA length
- longer DNA = Inc TM

TM depends on pH and ionic strength
- high salt content incr TM bc salt incr the stability of the DNA backbone

increases

Salt presence ______ the stability of the DNA backbone

1

Nitrogenous base on pentoses are found at position _____

5

Phosphate groups on pentoses are found at position _____

2

In DNA, there is no -OH group at position _____

phosphate

What provides the property of DNA: Pentose, Nitrogenous Base, or Phosphate?

DNA replication

Info from parental DNA copied to daughter DNA strand
- high fidelity

Transcription

RNA is synthesized using DNA as a template

DNA -> mRNA

1 strand of dsDNA acts as molecular template for RNA synthesis

Translation

Proteins synthesized based on info stored in ribonucleotide triplets in RNA

Triplets of nucleotides in mRNA bind to complementary triplets in tRNA

reverse transcription

synthesis of DNA from an RNA template
- viruses are able to perform this

ex) HIV, COVID-19

gene

defined as segments of DNA that code for peptides and RNA
- different from regulatory sequences
- DNA can be expressed differently to yield different products

tRNA

transfer RNA; type of RNA that carries amino acids to the ribosome
- utilized in translation

mRNA

messenger RNA; type of RNA that carries instructions from DNA in the nucleus to the ribosome
- complementary to DNA strand
- identical to non-template strand

25,000

The human genome contains around _________ genes

bacterial genomes

Genomes that contain:
- extrachromosomal, ds circular plasmids
- Around 2,000-20,000 bp long
- swapped easily between bacteria
- no essential genes, but often encode genes that degrade antibiotics
- plasmid exchange accounts for antibiotic resistance

plasmid exchange

One way bacteria can acquire antibiotic resistance

human genome

Genomes that:
- have 46 chromosomes (22 diploid pairs, plus X and Y)
- amounts to ~2 m of DNA
- length of each pair varies

mitochondria

This organelle has its own DNA separate from the eukaryote's genome
- codes for mitochondrial rRNAs, tRNAs, and some mitochondrial-specific proteins

"mtDNA"

Chromatin

Eukaryotic DNA that is organized with proteins (histones) into this complex

Noncoding sequences

most of the sequences that have no direct function (98% of human genome)
- some participate in regulation of gene expression (promoters, termination signals, etc.)
- some encode for small regulatory RNA
- some encode for unwanted genes or remnants of viral infections

microsatellite DNA

Repeated short simple sequence of DNA; the number of repeats is highly variable

genome-wide repeats

A large repetitive DNA sequence within the genome
- transposable elements

transposable elements

mobile pieces of DNA that can copy themselves into entirely new areas of the chromosomes

LINES

Long interspersed sequences

transposons

(jumping genes) short strands of DNA capable of moving from one location to another within a cell's genetic material
- ends contain terminal repeats that hybridize with complementary regions of target DNA during insertion
- account for ~50% of human genome

exons

expressed sequences of DNA translated into amino acid sequences
- account for only ~1.5% of human DNA
- spliced together after introns removed to create "mature transcripts"

introns

regions of genes that are transcribed but not translated
- they do not encode polypeptide sequence
- removed after transcription

bacterial introns

These introns:
- do not interrupt protein-coding sequences, only mainly interrupt tRNA sequences
- phage genomes within bacteria interrupt protein-coding sequences
- many encode catalytic RNA sequences that have the ability to insert and reverse transcribe themselves into genomic DNA

Simple Sequence Repeats (SSRs)

short sequences of ~10 bp or less that repeated millions of times within eukaryotic genomes
- aka. "Satellite" DNA
- associated with centromeres and telomeres

T

T/F: Eukaryotic chromosomes require centromeres, telomeres, and origin of replication to be maintained during cell division

centromeres

SSR Region where the 2 daughter chromosomes are held together during mitosis
- after DNA replication but before cell division

essential for equal distribution of chromosome sets to daughter cells

Have AT-rich repeated sequences of ~130 bp

AT

Centromeres have what nitrogenous base-pairs repeated in its sequence: AT or CG?

telomeres

SSR region that caps the end of eukaryotic chromosomes added by telomerase
- forms special loop structures to keep DNA ends from unraveling
- associated with cellular aging due to shortening after round of replication

Contain multiple repeats within general sequence
(TxGy)n and (AxCy)n, where n=1500 or more in mammals

shortened

telomeres are ________ after each round of DNA replication, which is associated with cellular aging

DNA supercoiling

the formation of additional coils in DNA due to twisting forces
- many circular DNAs do this
- has great influence on transcription and replication of DNA
- can be highly regulated

relaxed

Non-supercoiled DNA is also known as ________ DNA

Linking #

Invariant topological property of covalently closed, circular DNA (cccDNA) that is composed of Twist (T) and Writhe (W)

L = T + W

L can NEVER be changed as long as no topoisomerase is used and there are no nicks in the DNA

topoisomerase

Enzyme that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork.
- can change the linking # of circular DNA
- required for DNA unwinding and rewinding during transcription and replication

2 major types:
- Type I = make transient cut in ONE DNA strand, changes LK by 1 (changes twist #)

- Type II = make transient cut in BOTH DNA strands, changes LK in steps of 2 (changes Writhe #)

10.5

Normal B-form, relaxed DNA is _______ bp/turn

closed circular DNA

This DNA is:
- rarely relaxed
- strain induces supercoiling
- strain is due to fewer helical turns (underwinding)
- underwinding makes later separation of the strands easier

undwerwinding

__________ makes later separation of the strands easier

superhelical density

aka) Specific Linking Difference

In a helical molecule such as DNA, the number of supercoils (superhelical turns) relative to the number of coils (turns) in the relaxed molecule.

Sigma (o) = Delta LK / LK0

Ratio of # turns removed to LK0

For most cellular DNAs, o = -0.05 to -0.07

topoisomers

same DNA with different degrees of supercoiling
- conversions between these requires a DNA strand break

compact

Negatively supercoiled DNA means that the DNA is more: loose or compact?

negatively

________-supercoiled DNA travels faster in an agarose gel electrophoresis experiment that relaxed or nicked DNA does

Type I

Type of topoisomerase that makes a transient cut in ONE DNA strand, changing LK by 1
- changes the twist numbers

Type II

Type of topoisomerase that makes a transient cut in BOTH DNA strands, changing LK by steps of 2
- changes Writhe numbers

E. coli topoisomerases

Topo I and II are Type I:
- remove negative supercoils to relax DNA
- increases LK
- uses single-stranded breaks

Topo II is called DNA gyrase
- introduces negative supercoils
- decreases LK
- uses ATP and double-stranded breaks

DNA gyrase

aka) Topo II

- Introduces negative supercoils
- decreases LK
- uses ATP and double-stranded breaks
- relieves topological stress ahead of the replication forks

Topo II

Which E. coli topoisomerase uses ATP to introduce negative supercoils to DNA?

eukaryotic topoisomerases

Topo I and II are Type I

Type II topoisomerases include 2 subfamilies:
- Type IIA and Type IIB
- can relax both positive and negative supercoils

antibiotics

Topoisomerases are targets for _______, such as Coumarins or Quinolones

Coumarins

antibiotics that inhibit bacterial Type II topoisomerases from binding ATP

ex) Novobiocin, Coumermycin A1

Quinolones

antibiotics that inhibit the last step of resealing DNA strand breaks
- wide-spectrum and mostly selectve for bacterial enzymes

ex) Nalidixic acid; ciproflaoxadin, Cipro

inhibitors

Topoisomerase _________ are used as antibiotics

Topoisomerase inhibitors

These inhibitors target cancer cells bc most rapidly growing cells (tumors, others) express topoisomerases

Eukaryotic Type I
- captothecin, irinotecan (Campto), topotecan (Hycamtin)

Eukaryotic Type II
- doxorubicin (Adriamycin), etoposide (Etopophos), ellepticine

eukaryotic Type I topoisomerase inhibitors

trap the enzyme-DNA complex in its cleaved state
- ex) Captothecin, irinotecan (Campto),
topotecan (Hycamtin)

Eukaryotic Type II topoisomerase inhibitors

- Doxorubicin (Adriamycin), etoposide (Etopophos), ellepticine

G0

A nondividing state occupied by cells that have left the cell cycle, sometimes reversibly.
- chromatin is amorphous and randomly dispersed

Interphase

period of the cell cycle between cell divisions: G1, S, and G2 phases
- chromatin is amorphous and randomly dispersed

prophase of mitosis

chromosomes become condensed, and pairs of sister chromatids form