Molecular Biochemistry

Question 1

Describe the methylation and acetylation pattern of heterochromatin.

Answer 1

Heterochromatin is transcriptionally inactive (e.g. X chromosome Barr bodies) and are high methylated and less acetylated, resulting in a condensed structure.

Euchromatin is transcriptionally active and are highly acetylated and less methylated, resulting in a “looser” more accessible structure.

Question 2

What is the structure of chromatin?

Answer 2

Negatively charged DNA wraps twice around positively charged histones (rich in lysine and arginine) and further stabilized by H1 histones (“linker DNA”).

This enables formation of condensed DNA.

Question 3

Purines vs. pyrimidines?

Which bonds involve 3 H bonds and which involve 2 H bonds?

Which amino acids are necessary for de novo purine synthesis? Pyrimidine?

Answer 3

Purines - Adenine, guanine –> 2 rings
Pyrimidines - Cytosine, uracil, thymine (methylated vs. uracil) –> 1 ring

G-C bond = 2 hydrogen bonds (C-G bonds are like Crazy Glue)

GAG (amino acids) for purine synthesis - glycine, aspartate, glutamine

Aspartate for pyrimidine synthesis.

Question 4

How do each of the following drugs interfere with de novo pyrimidine/purine synthesis?

Answer 4

Inhibition of pyrimidine synthesis:

1) Leflunomide: inhibits dihydroorotate dehydrogenase
2) Methoxetrate, trimethoprim (TMP), pyrimethamine: inhibits dihydrofolate reductase
3) 5-FU (5-fluorouracil): inhibits thymidylate synthase

Inhibition of purine synthesis:

1) 6-MP (6-mercaptopurine): 6-MP + prodrug azathioprine
2) Mycophenolate and ribavirin: inhibits IMP dehydrogenase

Inhibition of pyrimidine AND purine synthesis:
1) Hydroxyurea: inhibits ribonucleotide reductase

First Aid, page 33

Question 5

Adenosine Deaminase Deficiency (ADA)

Answer 5

ADA required for degradation of adenosine and deoxyadenosine. Deficiency results in build up of toxic levels of product –> toxic to LYMPHOCYTES –> one major cause of autosomal recessive SCID.

First Aid, page 34

Question 6

Lesch Nyhan Syndrome - pathophysiology, clinical findings, treatment?

Answer 6

X-linked recessive defect in HGPRT in purine salvage pathway that converts hypoxanthine –> IMP and guanine –> GMP.

Rx: Allopurinol, Febuxostat (second line)

Clinical features:
Hyperuricemia - orange “sand,” sodium urate crystals
Gout - excess uric acid –> uric acid crystals
Pissed off - self mutilation, aggression
Retardation - intellectual disability
dysTonia

First Aid, page 35

Question 7

Function of DNA polymerase III and what mediates its action? What is the common mechanism of action for drugs acting on this enzyme?

Answer 7

DNA polymerase III is a prokaryotic polymerase that elongates leading. Cannot initiate replication (5’ –> 3’) until formation of RNA primer by primase.

Also has 3’ –> 5’ exonuclease proofreading activity.

Drugs often block replication via chain termination by modifying 3’ OH to prevent addition of next nucleotide.

First Aid, page 35

Question 8

DNA polymerase I

Answer 8

Prokaryote ONLY polymerase that has 5’ –> 3’ exonuclease activity to remove RNA primer and to replace it with nucleotides in 5’–> 3’ fashion.

First Aid, page 35

Question 9

DNA ligase

Answer 9

Catalyzes formation of phosphodiester bonds to join Okazaki fragments

First Aid, page 35

Question 10

Transition vs. tranversion point mutations (silent, missense, nonsense)

Answer 10

Transition - purine to purine, pyrimidine to pyrimidine
Tranversion - purine to pyrimidine, vice versa

First Aid, page 36

Question 11

What type of mutation describes that of sickle cell disease (glutamic acid –> valine)?

Answer 11

Missense mutation.

If new amino acid is structurally/chemically similar, then called “conservative” missense mutation.

First Aid, page 36

Question 12

Role of DNA topoisomerases? Mechanism of action for the following group of medications:

1) Irinotecan/topotecan
2) Etoposide/teniposide
3) Fluoroquinolones

Answer 12

DNA topoisomerases create a single or double stranded break in the helix to add or remove supercoils.

1) Irinotecan/topotecan - inhibit eukaryotic topoisomerase I
2) Etoposide/teniposide - inhibit eukaryotic topoisomerase II
3) Fluoroquinolones - inhibit prokaryotic topoisomerase II (DNA gyrase) and topoisomerase IV

First Aid, page 35

Question 13

What is the role and mechanism of nucleotide excision repair? Which phase of the cell cycle does it occur?

Answer 13

Single stranded-DNA repair mechanism

Nucleotide excision repair helps repair bulky, helix-distorting lesions in DNA, such as the formation of pyrimidine dimers in xeroderma pigmentosum upon exposure to UV light.

Specific endonucleases excise oligonucleotides containing damaged bases, with subsequent DNA polymerase and ligase filling in and resealing the gap.

Occurs in G1 phase of cell cycle.

First Aid, page 37

Question 14

What is the role and mechanism of base excision repair? Which phase of the cell cycle does it occur?

Answer 14

Single stranded-DNA repair mechanism

Base excision repair is important in repair of spontaneous/toxic deamination.

“GEL PLease” Mechanism of Action:

1) Base-specific Glycosylase removes altered base to create AP (apurinic/apyrimidinic site).
2) AP-Endonuclease removes one or more nucleotide(s) at 5’ end
3) Lyase cleaves 3’ end
4) DNA Polymerase-beta fils the gap
5) DNA Ligase seals gap

Occurs throughout cell cycle.

First Aid, page 37

Question 15

What is the role and mechanism of mismatch repair? Which phase of the cell cycle does it occur?

Answer 15

Single stranded-DNA repair mechanism

Used to repair/remove mismatched nucleotides of NEWLY synthesized strand.

Occurs during G2 phase of cell cycle.

First Aid, page 37

Question 16

What is the role and mechanism of nonhomologous end joining (NHEJ)?

Answer 16

Double stranded-DNA repair mechanism

Repairs double-stranded breaks by bringing together 2 ends of DNA fragments with NO HOMOLOGY IN DNA REQUIRED. As a result, some DNA may be lost.

Defective in ataxia telengiectasia, breast/ovarian cancers with BRCA1 mutations, Fanconi anemia.

First Aid, page 37

Question 17

How do the mRNA start codons for eukaryotes and prokaryotes differ?

Answer 17

Eukaryotic start codon AUG codes for methionine, which may be removed before translation is completed.

In contrast, AUG codes for N-formylmethionine (fMet) in prokaryotes. fMet stimulates neutrophil chemotaxis.

First Aid, page 37

Question 18

Describe the role of each of these sites of gene expression regulators:

1) Promoters
2) Enhancers
3) Silencers

Answer 18

1) Site upstream from gene locus where RNA polymerase II and other transcription factors bind. Example: TATA box
2) Stretch of DNA that alters gene expression by binding transcription factors like activator proteins.
3) Site where negative regulators like repressors bind.

First Aid, page 38

Question 19

Compare RNA polymerases in eukaryotes vs. prokaryotes.

Answer 19

Eukaryotes have 3 types of RNA polymerases:

1) RNA polymerase I - makes rRNA
2) RNA polymerase II - makes mRNA –> initiates transcription by opening DNA at promoter site; inhibited by alpha-amanitin in death cap mushrooms (Amanita phalloides)
3) RNA polymerase III - makes tRNA

Prokaryotes have ONLY ONE type of RNA polymerase that makes all 3 kinds of RNA. Inhibited by rifampin.

Actinomycin-D inhibits both prokaryotic and eukaryotic RNA polymerase.

First Aid, page 38

Question 20

What are the steps of RNA processing necessary in order to generate mRNA?

Answer 20

Initial transcript is called heterogeneous nuclear RNA (hnRNA), which then undergoes three modification processes:

1) 5’ 7-methylguanosine cap
2) 3’ polyadenylation - NO template required for poly-A polymerase
3) Splicing out introns

mRNA is then transported out of nucleus and into cytosol for translation.

First Aid, page 38

Question 21

What are P-bodies/cytoplasmic processing bodies?

Answer 21

Site of mRNA quality control and consist of exonucleaes, decapping enzymes, and microRNAs.

mRNAs may also be stored here for future translation.

First Aid, page 38

Question 22

What are the factors and mechanism involved with splicing? Name some autoimmune diseases associated with the splicing machinery.

Answer 22

1) Primary transcript combines with snRNPs and other proteins to form spliceosome.
2) Lariat intermediate formed and released to remove intron. Exons join.

SLE - anti-Smith are antibodies to spliceosome snRNPs
MCTD - Anti-U1 RNP antibodies

First Aid, page 39

Question 23

Role of microRNAs?

Answer 23

Small, conserved non-coding RNA molecules that POST-TRANSCRIPTIONALLY regulate gene expression.

Target 3’ untranslated region of mRNAs to either degrade or repress translation.

First Aid, page 39

Question 24

Describe the structure of tRNA, especially each of the following components:

1) Anticodon
2) Aminoacyl
3) T-arm
4) D-arm

Answer 24

1) Anticodon is opposite of aminoacyl end. It is the part that binds/complements the mRNA transcript.
2) Aminoacyl 3’ end consists of CCA sequence and covalently binds the amino acid during translation.
3) T-arm contains TpsiC (ribothymidine, pseudouridine, cytidine) sequence necessary for ribosome binding.
4) D-arm contains dihydrouridine residues necessary for tRNA recognition by aminoacyl tRNA synthetase.

First Aid, page 40

Question 25

Steps and factors involved with initiation of translation? Eukaryotic vs. prokaryotic ribosomal subunits?

Answer 25

Initiation begins with GTP hydrolysis. (Eukaryotic) initiation factors then help assemble 40S ribosomal subunit with initiator tRNA, which then is released when mRNA and ribosomal 60S subunit assemble with complex (80S complete unit).

Prokaryotic ribosomal subunits are 30S and 50S (70S complete unit).

First Aid, page 41

Question 26

Steps and factors involved with elongation of translation? What are the different binding sites of the ribosomal subunit?

Answer 26

APE binding sites:
A - aminoacyl-tRNA binding site
P - growing peptide
E - empty tRNA as it exits

1) Aminoacyl-tRNA binds to A site EXCEPT for initiator methionine.
2) rRNA acts as a ribozyme and catalyzes peptide bond formation and transfers growing polypeptide to amino acid in A site
3) Ribosome advances 3 nucleotides toward 3’ end of mRNA to move peptidyl tRNA to P site (translocation).

First Aid, page 41

Question 27

Steps and factors involved with termination of translation?

Answer 27

Stop codon recognized by release factor and polypeptide released from ribosome.

First Aid, page 41