Nucleic Acid, Mutation, Chromatin, and Gene Expression

Two Main Classes of Nucleic Acids

1. ______ —> primarily present in the nucleus

2. ______ —> synthesized in the nucleus and then translocated in the cytoplasm

1. DNA

2. RNA

______ are building blocks of nucleic acids

nucleotides

______ —> nucleotide without the phosphate

nucleoside

each nucleotide has 3 components…

a sugar, a phosphate group, and a nitrogenous base.

nucleic acids (DNA vs RNA) differ based on their sugar:

1. ______ —> ribose sugar, which has a hydroxyl group (-OH) on the 2’ carbon —> more prone to hydrolysis —> LESS stable

2. ______ —> deoxyribose sugar, which lacks that oxygen (it has -H on the 2’ carbon

1. RNA

2. DNA

Nitrogenous Bases:

• ______ (2 rings, larger bases)

  • Adenine (A)

  • Guanine (G)

• ______ (1 ring, smaller bases)

  • Cytosine (C)

  • Thymine (T) (DNA only)

  • Uracil (U) (RNA only, replaces T)

• Purines

• Pyrimidines

Base Pairing Rules

______: A ↔ T, G ↔ C

______: A ↔ U, G ↔ C

• DNA

• RNA

Nitrogenous bases link to the sugar backbone at the 1′ carbon through a ______ bond, forming the nucleoside structure (base + sugar) —> replacing the -OH group at this position

glycosidic

Phosphate Group

• phosphate group contains one or two negatively charged ______ atom

• negative charge helps in the ______ of proteins to DNA

• negative charge allows DNA to remain ______ when denatured because negative charges repel themselves

• oxygen

• binding

• straight

Nucleic Acid

1. alternating ______ + ______ = DNA/RNA backbone

2. phosphate connects the 3′ carbon of one sugar to the 5′ carbon of the next sugar —> forms two ester bonds —> ______ ______

3. Nucleic acids always have two ends:

   • 5′ end = has a free ______ group

   • 3′ end = has a free ______ group

1. sugar, phosphate

2. phosphodiester bond

3. phosphate

4. -OH

1. ______ bonds —> between nitrogenous bases —> weakest bonds

2. ______ bonds —> between the sugar and a base

3. ______ bonds —> between the sugar and a phosphate

1. hydrogen

2. glycosidic

3. phosphodiester

1. DNA is double-stranded —> the ______ orientation of DNA allows for the base pairs to compliment one another

2. DNA is double helix —> the two helical strands are connected through ______ bonds between pairs of nucleotides

1. antiparallel

2. hydrogen

1. ______ hydrogen bonds between G and C —> stronger DNA

2. ______ hydrogen bonds between A and T

1. three

2. two

1. ______ ______ —> is essential for several biological processes including DNA compaction, DNA metabolism, and likely gene expression

two types:

1. ______ supercoiling —> more coiling —> tighter compaction

2. ______ supercoiling —> DNA unwinds —> relaxed form

1. DNA supercoiling

2. positive

3. negative

1. ______ —> enzymes that monitor and adjust DNA supercoiling by…

   • create temporary strand ______ in DNA

   • ______ and ______ DNA strands

   • allow ______ to interact with DNA

   • prevent ______ supercoiling, which can damage DNA

• topoisomerases

• cuts

• uncoil, reseal

• proteins

• excessive

Two Main Types of Topoisomerases

1. type ______ topoisomerases —> cut one strand of DNA

2. type ______ topoisomerases —> cut two strands of DNA

1. one

2. two

Type I Topoisomerases MOA

• make a break in ______ DNA strand

• pass the other strand through the ______

• ______ the break

Effect —> DNA winding/unwinding without ATP use.

• one

• break

• reseal

Type II Topoisomerases MOA

1. Topo II binds to ______ ______.

2. Complex binds to ______ ______.

3. ______ binds and promotes formation of the topological complex.

4. ______ ______ of the G duplex.

5. ______ ______ passes through the gap.

6. The G duplex is ______ and the bound ______ hydrolyzed.

1. G (gate) duplex

2. T (transported) duplex

3. ATP

4. Mg²⁺-dependent cleavage

5. re-ligated, ATP

Topoisomerase inhibitors prevent DNA repair → this leads to DNA damage → triggers ______ ______ —> utilized as anti-______ and anti-______ agents.

cell death, cancer, bacterial

mRNA

• synthesized from ______ via ______

• serves as the link between ______ and ______

• codes for ______

• ______ and single-stranded RNA

• a ______ cap is added at the 5’ end of pre-mRNA while mRNA is still being made

• a ______ tail is added to the 3’ end of the pre-mRNA once mRNA synthesis is completed

• DNA, transcription

• proteins, DNA

• proteins

• unstable

• 7-methylguanosine

• poly (A)

rRNA

• ______ RNA molecules

• comprising ______% of ribosome’s mass

• ensures the proper ______ of mRNA, tRNA, and ribosome during protein synthesis

• stable

• 60

• alignment

tRNA

• ______ RNA with extensive intramolecular base pairing

• contains an ______ ______ binding site and an ______ binding site

• carries the correct animo acid to the site of protein synthesis in the ______

• stable

• amino acid, mRNA

• ribosome

Replication = DNA → ______

Why it’s essential: 

—> when a cell divides, each ______ cell must receive the exact same genetic information as the parent cell.

Sequence of Events:

1. ______ is replicated.

2. ______ divides.

3. Both new daughter cells contain ______ DNA.

• DNA

• daughter

• DNA
  cell

• identical

• Both parental DNA strands serve as ______ for making new DNA.

• ______ model —> each DNA molecule has (1) 1 parental (old) strand and (2) 1 newly synthesized strand.

• templates

• semiconservative

Stages of DNA Replication

1. ______ —> the DNA helix is opened up and the DNA replication proteins are positioned

2. ______ —> synthesis of the new DNA strands

3. ______ —> DNA synthesis is stopped

1. initiation

2. elongation

3. termination

DNA replication begins at the ______ of replication where two ______ ______ are formed —> DNA synthesis proceeds ______

origin, replication forks, bidirectionally

• ______ DNA has a single replication origin site

• ______ DNA has multiple origins of replication

• bacterial

• eukaryotic

Central Dogma

1. replication: DNA → DNA

2. transcription: DNA → RNA

3. translation: RNA → protein

DNA Replication —> Initiation

• ______ unwinds the DNA helix —> causes ______ ______ of the DNA

• ______ prevent the topological distortion by making temporary single-strand cuts in the DNA —> later resealed

• ______-______ DNA binding proteins (SSB) prevents the two original strands from re-forming a double stranded molecule

• helicase, topological distortion

• topoisomerase 1

• single-stranded

DNA Replication —> Elongation

1. ______ —> generates short RNA strand that binds to the single-stranded DNA to initiate DNA synthesis

2. DNA polymerase reads the parental strand _____, but it synthesizes the new strand ______.

3. Adds ______ to the growing chain by forming ______ ______ at the 3’ end.

4. ______ strand —> continuous replication

5. ______ strand —> discontinuous replication in short fragments —> Okazaki fragments —> later linked together with the help of ______

1. primase

2. 3’ → 5’, 5’ → 3’

3. nucleotides, phosphodiester bonds

4. leading

5. lagging, ligase

A ______ of the template for the ______ strand places it in position for 5’ → 3’ polymerization —> enables DNA polymerase to synthesize both daughter strands simultaneously.

looping, lagging

DNA Replication Termination

1. Once DNA synthesis is completed, the RNA primer is removed by ______ ______

2. ______ ______ fills the gap where the RNA primer used to be

3. ______ ______ seals the last two nucleotides together

1. RNA hybridase

2. DNA polymerase

3. DNA ligase

proofreading —> ______ ______ —> if DNA polymerase detects the mis-paired deoxynucleotides —> DNA polymerase shifts backward ( ______ direction) —> removes incorrect base —> addition of correct base

exonuclease activity, 3’ → 5’

DNA Polymerase Inhibitors in Cancer Therapy

1. Gemcitabine —> a ______ ______ —> a “fake nucleotide” —> incorporated into replicating DNA —> inhibits ______ ______ —> stalls the ______ ______ —> cancer cells can’t divide —> leads to cell death.

2. used to treat ______, ______, and ______ cancers

1. nucleoside analog, DNA polymerase, replication fork

2. pancreatic, lung, bladder

DNA polymerases cannot fully ______ the very ends of the chromosome —> with each replication round, a small portion of the DNA is lost at the 3’ ends —> a ______ DNA sequence is added after replication to protect the ends of chromosomes

replicate, telomere

______ is a reverse transcriptase that uses an RNA molecule as a template —> adds repetitive DNA sequence of ______ nucleotide after replication

telomerase, six

Cells with High Telomerase Activity (3)

1. white blood cells

2. stem cells

3. cancer cells

• healthy somatic cells have very little or no telomerase activity

• cells that need to proliferate have high ______ activity

• ______ telomerase = unlimited division potential (stem cells, immune cells, cancers)

• ______ telomerase = limited division capacity (normal body cells → aging)

• telomerase

• high

• low

telomere ______ decreases with age in proliferating tissues —> progressive shortening of telomeres leads to —> (3)

length, metabolic arrest, cell death, oncogenic transformation

telomere damage or deletion leads to loss of ______ ______/______ —> characteristic feature of ______

genome integrity/stability, cancer

• telomerase is significantly over expressed in 80-95% of all ______ tumors

• ______ —> present in green tea —> a natural telomerase inhibitor

• malignant

• epigallocatechin-3-gallate 

• ______ = just the double helix

• ______ = DNA wrapped around structural proteins

• chromatin made by ______ proteins

• chromatin is further condensed to form ______

• DNA

• chromatin

• histones

• chromosomes

Chromatin exists in two forms:

• ______ —> loosely packed, lightly stained —> active

• ______ —> densely packed, darkly stained —> inactive

• euchromatin

• heterochromatin

Features of Mitochondrial DNA (4)

1. circular

2. lacks structural proteins

3. lacks telomeres

4. less base pairs (16,500 vs. 3.2 billion in nuclear DNA)

Gene Expression —> Two Major Steps

1. ______ —> DNA —> mRNA in the nucleus

2. ______ —> mRNA —> proteins in the cytoplasm

1. transcription

2. translation

Out of the 3 billion base pairs in the human genome:

• ______% —> genes —> proteins

• ______% —> non-coding DNA

• 2

• 98

Transcription Steps

1. ______ —> RNA polymerase is positioned on the DNA of the gene that needs to be transcribed

2. ______ —> RNA polymerase transcribes the DNA sequence of the gene into an RNA molecule

3. ______ —> RNA polymerase is released from the DNA

1. initiation

2. elongation

3. termination

Transcription —> Initiation

• each gene has a ______

• the general ______ ______ bind to the promoter

• ______ ______ attaches to the transcription factors forming a transcription initiation complex

• promoter

• transcription factors

• RNA polymerase

Transcription —> Elongation

1. RNA polymerase moves along one strand of DNA called the ______ strand in the ______ direction.

2. The strand of DNA not used as a template for transcription is called the ______ strand.

3. The newly synthesized mRNA has the same nucleotide sequence as the ______ strand.

1. template/non-coding, 3’ → 5’

2. non-template/coding

3. coding

Transcription —> Termination

• not well understood in eukaryotes

• RNA polymerase transcribes a ______ ______: AAUAAA —> called the ______ signal.

• Once the poly-A signal is synthesized, mRNA is ______ and ______ ______ adds about ~200 adenine nucleotides to the 3’ end —> creates the poly-A tail.

• consensus sequence, poly-A

• cleaved, poly(A) polymerase

Precursor mRNA (pre-mRNA) in Eukaryotes

• RNA transcript = ______ —> must be processed into a ______ before leaving the nucleus

• a ______ is added to the 5’ end

• a ______ is added to the 3’ end

• pre-mRNA, mRNA

• cap

• poly-A tail

Processing of eukaryotic pre-mRNA: RNA splicing

• Pre-mRNA has both:

  • ______ = coding segments (kept)

  • ______ = noncoding segments (removed)

• ______ —> splice out introns and combine exons via spliceosome that contains small nuclear RNAs and proteins

• exons

• introns

• RNA splicing

• ______ = a set of 3 consecutive bases in mRNA that code for 1 amino acid.

• read in the ______ direction of the mRNA

• each codon ______ an amino acid

• ______ “stop” codons mark the end of a protein

• ______ “start” codon —> ______ —> marks the beginning of a protein

• codons specify the order of amino acids in a protein from ______ (methionine) to ______

• human genetic codes = ______ amino acids —> ______ codons

• codon

• 5’ → 3’

• specify

• three

• one, AUG

• N-terminus → C-terminus

• 20, 64

______ RNA polymerase is a promising target for the discovery of new antimicrobial agents

bacterial

Reverse Transcription ( ______ → ______ )

• RNA virus = ______ —> makes a complementary DNA ( ______ ) —> integrates into the host’s genome

• ______ ______ makes cDNA from RNA

• ^ inhibitors are a class of ______ drugs used to treat HIV infections or AIDS

• RNA → DNA

• retrovirus, cDNA 

• reverse transcriptase

• antiretroviral

Ribosomes

• sites in a cell in which ______ takes place

• made of a large and a small subunit which come together around a mRNA molecule —> complete ribosome

• ribosomes are 1/3 ______ and 2/3 ______ ______

• sites for tRNA binding

  • ____ site —> peptidyl or donor site

  • ____ site —> aminoacyl or acceptor site

  • ____ site —> exit site

• translation

• proteins, ribosomal RNA (rRNA)

• P

• A

• E

Translation involves 3 steps…

1. initiation

2. elongation

3. termination

Translation —> Initiation 

1. the initiator tRNA carrying _______ attaches to the small ribosomal subunit

2. the complex “walk” along the mRNA in the ____ direction to search for the start codon ______

3. the large ribosomal subunit joins to form ______ complex at the site of AUG on mRNA

1. methionine

2. 3’, AUG

3. initiation

Translation —> Elongation

1. ______-______ tRNA starts in the ______ site of the ribosome

2. ______ then binds to ______ site

3. ______ ______ connects amino acid to one another —> mRNA pulled onward through the ribosome by exactly one codon

1. methionine-carrying, P site

2. aminoacyl tRNA, A

3. peptide bonds

Translation —> Termination

• happens when a ______ codon in the mRNA enters the _____ site

• stop codons are recognized by ______ ______ which fit neatly into the A site of ribosome

• upon termination, the ribosome is disassembled and the completed polypeptide is released

• stop, A

• release factors

Effects of DNA Mutations on Protein Synthesis

• A different protein may form

• Protein may not form at all

• Less amount of protein may form

• Protein production may increase

• May not affect protein production