Biochemistry - Molecular

Question 1

Chromatin structure

• DNA
• Histones

Answer 1

• DNA
  
  • DNA exists in the condensed, chromatin form in order to fit into the nucleus.
  • Negatively charged DNA loops twice around positively charged histone octamer to form nucleosome “bead.”
    
    • Think of “beads on a string.”
  • In mitosis, DNA condenses to form chromosomes.
  • DNA and histone synthesis occur during S phase.
• Histones
  
  • Histones are rich in the amino acids lysine and arginine.
  • H1 binds to the nucleosome and to “linker DNA,” thereby stabilizing the chromatin fiber.
  • H1 is the only histone that is not in the nucleosome core

Question 2

Heterochromatin

Answer 2

• Condensed, transcriptionally inactive, sterically inaccessible.
• HeteroChromatin = Highly Condensed.

Question 3

Euchromatin

Answer 3

• Less condensed, transcriptionally active, sterically accessible.
• Eu = true, “truly transcribed.”

Question 4

DNA methylation

Answer 4

• Template strand cytosine and adenine are methylated in DNA replication, which allows mismatch repair enzymes to distinguish between old and new strands in prokaryotes.
• DNA methylation at CpG islands represses transcription.
• CpG Methylation Makes DNA Mute.

Question 5

Histone methylation

Answer 5

• Usually reversibly represses DNA transcription, but can activate it in some cases.
• Histone Methylation Mostly Makes DNA Mute.

Question 6

Histone acetylation

Answer 6

• Relaxes DNA coiling, allowing for transcription.
• Histone Acetylation makes DNA Active.

Question 7

Nucleotides

• Purines
• Pyrimidines
• Thymine
• Uracil
• Bonds
• Amino acids necessary for purine synthesis
• Nucleosides
• Nucleotides

Answer 7

• Purines
  
  • Purines (A, G)—2 rings.
  • PURe As Gold
• Pyrimidines
  
  • Pyrimidines (C, T, U)—1 ring.
  • CUT the PY (pie)
• Thymine
  
  • Has a methyl.
    
    • THYmine has a meTHYl
  • Found in DNA
• Uracil
  
  • Deamination of cytosine makes uracil.
  • Found in RNA
• Bonds
  
  • G-C bond (3 H bonds) stronger than A-T bond (2 H bonds). 
  • Increased G-C content –> increased melting temperature of DNA.
• Amino acids necessary for purine synthesis (GAG)
  
  • Glycine
  • Aspartate
  • Glutamine
• Nucleosides
  
  • NucleoSides = base + (deoxy)ribose (Sugar).
• ​Nucleotides
  
  • NucleoTides = base + (deoxy)ribose + phosphaTe
  • Linked by 3′-5′ phosphodiester bond.

Question 8

De novo pyrimidine and purine synthesis

• Purines
• Pyrimidines
• Ribonucleotides
• Carbamoyl phosphate

Answer 8

• Purines
  
  • Start with sugar + phosphate (PRPP)
  • Add base
• Pyrimidines
  
  • Make temporary base (orotic acid)
  • Add sugar + phosphate (PRPP)
  • Modify base
• Ribonucleotides
  
  • Synthesized first
  • Converted to deoxyribonucleotides by ribonucleotide reductase.
• Carbamoyl phosphate
  
  • Involved in 2 metabolic pathways: de novo pyrimidine synthesis and the urea cycle.

Question 9

Various antineoplastic and antibiotic drugs function by interfering with nucleotide synthesis:

• Leflunomide
• ƒƒMycophenolate and ribavirin
• ƒƒHydroxyurea
• 6-mercaptopurine (6-MP) and its prodrug azathioprine
• 5-fluorouracil (5-FU)
• ƒƒMethotrexate (MTX), trimethoprim (TMP), and pyrimethamine

Answer 9

• Leflunomide
  
  • Inhibits dihydroorotate dehydrogenase
• ƒƒMycophenolate and ribavirin
  
  • Inhibit IMP dehydrogenase
• ƒƒHydroxyurea
  
  • Inhibits ribonucleotide reductaseƒƒ
• 6-mercaptopurine (6-MP) and its prodrug azathioprine
  
  • Inhibit de novo purine synthesis
• 5-fluorouracil (5-FU)
  
  • Inhibits thymidylate synthase (decrease deoxythymidine monophosphate [dTMP])
• ƒƒMethotrexate (MTX), trimethoprim (TMP), and pyrimethamine
  
  • Inhibit dihydrofolate reductase (decrease dTMP) in humans, bacteria, and protozoa, respectively

Question 10

Purine salvage deficiencies

• HGPRT + PRPP
• APRT + PRPP
• Adenosine deaminase (ADA)
• Xanthine oxidase

Answer 10

• HGPRT + PRPP [1]
  
  • Guanine –> Guanylic acid (GMP)
  • Hypoxanthine –> Inosinic acid (IMP)
• APRT + PRPP [2]
  
  • Adenine –> Adenylic acid (AMP)
• Adenosine deaminase (ADA) [3]
  
  • Adenosine –> Inosine
• Xanthine oxidase [4]
  
  • Hypoxanthine –> Xanthine
  • Xanthine –> Uric acid

Question 11

Adenosine deaminase deficiency

Answer 11

• Excess ATP and dATP imbalances nucleotide pool via feedback inhibition of ribonucleotide reductase –>Ž prevents DNA synthesis and thus decreases lymphocyte count.
• One of the major causes of autosomal recessive SCID.

Question 12

Lesch-Nyhan syndrome

• Definition
• Findings
• ​Treatment

Answer 12

• Definition
  
  • Defective purine salvage due to absent HGPRT, which converts hypoxanthine to IMP and guanine to GMP.
  • Results in excess uric acid production and de novo purine synthesis.
  • X-linked recessive.
• Findings
  
  • Intellectual disability, self-mutilation, aggression, hyperuricemia, gout, dystonia.
  • HGPRT:
    
    • Hyperuricemia
    • Gout
    • Pissed off (aggression, self-mutilation)
    • Retardation (intellectual disability)
    • DysTonia
• ​Treatment
  
  • Allopurinol or febuxostat (2nd line).

Question 13

Genetic code features

• Unambiguous
• Degenerate / redundant
• Commaless, nonoverlapping
• Universal

Answer 13

• Unambiguous
  
  • Each codon specifies only 1 amino acid.
• Degenerate / redundant
  
  • Most amino acids are coded by multiple codons.
  • Exceptions: methionine and tryptophan encoded by only 1 codon (AUG and UGG, respectively).
• Commaless, nonoverlapping
  
  • Read from a fixed starting point as a continuous sequence of bases.
  • Exceptions: some viruses.
• Universal
  
  • Genetic code is conserved throughout evolution.
  • Exception in humans: mitochondria.

Question 14

DNA replication:
Eukaryotic vs. Prokaryotic

Answer 14

• Eukaryotic DNA replication is more complex than the prokaryotic process but uses many analogous enzymes.
• In both prokaryotes and eukaryotes, DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis.

Question 15

DNA replication (69)

• Origin of replication
• Replicaiton fork
• Helicase
• Single-stranded binding proteins
• DNA topoisomerases
• Primase

Answer 15

• Origin of replication [A}
  
  • Particular consensus sequence of base pairs in genome where DNA replication begins.
  • May be single (prokaryotes) or multiple (eukaryotes).
• Replicaiton fork [B]
  
  • Y-shaped region along DNA template where leading and lagging strands are synthesized.
• Helicase [C]
  
  • Unwinds DNA template at replication fork.
• Single-stranded binding proteins [D]
  
  • Prevent strands from reannealing.
• DNA topoisomerases [E]
  
  • Create a single- or double-stranded break in the helix to add or remove supercoils.
  • Fluoroquinolones—inhibit DNA gyrase (prokaryotic topoisomerase II).
• Primase [F]
  
  • Makes an RNA primer on which DNA polymerase III can initiate replication.

Question 16

DNA replication (69)

• DNA polymerase III
• DNA polymerase I
• DNA ligase
• Telomerase

Answer 16

• DNA polymerase III [G]
  
  • Prokaryotic only.
  • Elongates leading strand by adding deoxynucleotides to the 3′ end.
  • Elongates lagging strand until it reaches primer of preceding fragment.
  • 3′ –>Ž 5′ exonuclease activity “proofreads” each added nucleotide.
  • DNA polymerase III has 5′ Ž–> 3′ synthesis and proofreads with 3′ –>Ž 5′ exonuclease.
• DNA polymerase I [H]
  
  • Prokaryotic only.
  • Degrades RNA primer; replaces it with DNA.
  • Has same functions as DNA polymerase III but also excises RNA primer with 5′ Ž–> 3′ exonuclease.
• DNA ligase [I]
  
  • Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA (i.e., joins Okazaki fragments).
  • Seals.
• Telomerase
  
  • An RNA-dependent DNA polymerase that adds DNA to 3′ ends of chromosomes to avoid loss of genetic material with every duplication.

Question 17

Mutations in DNA

• Severity of damage (increasing)
• For silent, missense, and nonsense mutations:
  
  • Transition
  • Transversion

Answer 17

• Severity of damage (increasing)
  
  • Silent << missense < nonsense < frameshift.
• For silent, missense, and nonsense mutations:
  
  • Transition
    
    • Purine to purine (e.g., A to G) or pyrimidine to pyrimidine (e.g., C to T).
  • Transversion
    
    • Purine to pyrimidine (e.g., A to T) or pyrimidine to purine (e.g., C to G).

Question 18

Mutations in DNA

• Silent
• Missense
• Nonsense
• Frameshift

Answer 18

• Silent
  
  • Nucleotide substitution but codes for same (synonymous) amino acid
  • Often base change in 3rd position of codon (tRNA wobble).
• Missense
  
  • Nucleotide substitution resulting in changed amino acid
  • Called conservative if new amino acid is similar in chemical structure.
  • Ex. Sickle cell disease
• Nonsense
  
  • Nucleotide substitution resulting in early stop codon.
  • Stop the nonsense!
• Frameshift
  
  • Deletion or insertion of a number of nucleotides not divisible by 3, resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein.
  • Ex. Duchenne muscular dystrophy

Question 19

Nucleotide excision repair

• Single vs. double strand
• Definition
• Pathology

Answer 19

• Single vs. double strand
  
  • Single strand DNA repair
• Definition
  
  • Specific endonucleases release the oligonucleotide-containing damaged bases.
  • DNA polymerase and ligase fill and reseal the gap, respectively.
  • Repairs bulky helix-distorting lesions.
• Pathology
  
  • Defective in xeroderma pigmentosum, which prevents repair of pyrimidine dimers because of ultraviolet light exposure.

Question 20

Base excision repair

• Single vs. double strand
• Definition

Answer 20

• Single vs. double strand
  
  • Single strand DNA repair
• Definition
  
  • Base-specific glycosylase recognizes altered base and creates AP site (apurinic/apyrimidinic).
  • One or more nucleotides are removed by APendonuclease, which cleaves the 5′ end.
  • Lyase cleaves the 3′ end.
  • DNA polymerase-β fills the gap and DNA ligase seals it.
  • Important in repair of spontaneous/toxic deamination.

Question 21

Mismatch repair

• Single vs. double strand
• Definition
• Pathology

Answer 21

• Single vs. double strand
  
  • Single strand DNA repair
• Definition
  
  • Newly synthesized strand is recognized, mismatched nucleotides are removed, and the gap is filled and resealed.
• Pathology
  
  • Defective in hereditary nonpolyposis colorectal cancer (HNPCC).

Question 22

Nonhomologous end joining

• Single vs. double strand
• Definition
• Pathology

Answer 22

• Single vs. double strand
  
  • Double strand DNA repair
• Definition
  
  • Brings together 2 ends of DNA fragments to repair double-stranded breaks.
  • No requirement for homology.
• Pathology
  
  • Mutated in ataxia telangiectasia.

Question 23

DNA/RNA/protein synthesis direction

• DNA and RNA
• Protein synthesis
• mRNA
• 3’ hydroxyl attack

Answer 23

• DNA and RNA
  
  • Both synthesized 5′ –>Ž 3′.
  • The 5′ end of the incoming nucleotide bears the triphosphate (energy source for bond).
• Protein synthesis
  
  • N-terminus to C-terminus.
• mRNA
  
  • Read 5′ to 3′.
• 3’ hydroxyl attack
  
  • The triphosphate bond is the target of the 3′ hydroxyl attack.
  • Drugs blocking DNA replication often have modified 3′ OH, preventing addition of the next nucleotide (“chain termination”).

Question 24

Start and stop codons

• mRNA start codons
• Eukaryotic vs. prokaryotic start codons
• mRNA stop codons

Answer 24

• mRNA start codons
  
  • AUG (or rarely GUG).
  • AUG inAUGurates protein synthesis
• Eukaryotic vs. prokaryotic start codons
  
  • Eukaryotes: Codes for methionine, which may be removed before translaiton is completed.
  • Prokaryotes​: Codes for formylmethionine (f-met).
• mRNA stop codons
  
  • UGA, UAA, UAG
  • UGA = U Go Away.
  • UAA = U Are Away.
  • UAG = U Are Gone.

Question 25

Functional organization of a eukaryotic gene

Answer 25

Question 26

Regulation of gene expression

• Promoter
• Enhancer
• Silencer
• Locaiton of enhancers and silencers

Answer 26

• Promotor
  
  • Site where RNA polymerase and multiple other transcription factors bind to DNA upstream from gene locus (AT-rich upstream sequence with TATA and CAAT boxes).
  • Promoter mutation commonly results in dramatic decrease in level of gene transcription.
• Enhancer
  
  • Stretch of DNA that alters gene expression by binding transcription factors.
• Silencer
  
  • Site where negative regulators (repressors) bind.
• Locaiton of enhancers and silencers
  
  • May be located close to, far from, or even within (in an intron) the gene whose expression it regulates.

Question 27

Eukaryotic RNA polymerases

• What polymerases make
  
  • RNA polymerase I
  • RNA polymerase II
  • RNA polymerase III
• Functions
• Inhibition
• Prokaryotic polymerases

Answer 27

• What polymerases make
  
  • RNA polymerase I makes rRNA
    
    • Most numerous RNA, Rampant.
  • RNA polymerase II makes mRNA
    
    • Largest RNA, Massive.
  • RNA polymerase III makes tRNA
    
    • Smallest RNA, Tiny.
  • I, II, and III are numbered as their products are used in protein synthesis.
• Functions
  
  • No proofreading function, but can initiate chains.
  • RNA polymerase II opens DNA at promoter site.
• Inhibition
  
  • α-amanitin, found in Amanita phalloides (death cap mushrooms), inhibits RNA polymerase II.
  • Causes severe hepatotoxicity if ingested.
• Prokaryotic polymerases
  
  • 1 RNA polymerase (multisubunit complex) makes all 3 kinds of RNA.

Question 28

Eukaryotic RNA processing

• hnRNA
• The following processes occur in the nucleus following transcription:
• mRNA
• Poly-A polymerase
• Polyadenylation signal

Answer 28

• hnRNA
  
  • Initial transcript is called heterogeneous nuclear RNA (hnRNA).
  • hnRNA is then modified and becomes mRNA.
• The following processes occur in the nucleus following transcription:
  
  • Capping of 5′ end (addition of 7-methylguanosine cap)
  • Polyadenylation of 3′ end (≈ 200 A’s)
  • Splicing out of introns
• mRNA
  
  • Capped, tailed, and spliced transcript is called mRNA.
  • mRNA is transported out of the nucleus into the cytosol, where it is translated.
  • mRNA quality control occurs at cytoplasmic P-bodies, which contain exonucleases, decapping enzymes, and microRNAs
    
    • mRNAs may be stored here for future translation.
• Poly-A polymerase
  
  • Does not require a template.
• Polyadenylation signal
  
  • AAUAAA

Question 29

Splicing of pre-mRNA

• Splicing process
• Antibodies

Answer 29

• Splicing process
  
  1. Primary transcript combines with small nuclear ribonucleoproteins (snRNPs) and other proteins to form spliceosome.
  2. Lariat-shaped (looped) intermediate is generated.
  3. Lariat is released to precisely remove intron and join 2 exons.
• Antibodies
  
  • Antibodies to spliceosomal snRNPs (anti-Smith antibodies) are highly specific for SLE.
  • Anti-U1 RNP antibodies are highly associated with mixed connective tissue disease.

Question 30

Introns vs. Exons

• Introns
• Exons
• Abnormal splicing variants

Answer 30

• Introns
  
  • Introns are intervening noncoding segments of DNA.
  • Introns are intervening sequences and stay in the nucleus.
• Exons
  
  • Exons contain the actual genetic information coding for protein.
  • Different exons are frequently combined by alternative splicing to produce a larger number of unique proteins.
  • Exons exit and are expressed.
• Abnormal splicing variants
  
  • Implicated in oncogenesis and many genetic disorders (e.g., β-thalassemia).

Question 31

tRNA Structure

• Structure
• T-arm
• D-arm
• Acceptor stem

Answer 31

• Structure
  
  • 75–90 nucleotides, 2º structure, cloverleaf form, anticodon end is opposite 3′ aminoacyl end.
  • All tRNAs, both eukaryotic and prokaryotic, have CCA at 3′ end along with a high percentage of chemically modified bases.
  • The amino acid is covalently bound to the 3′ end of the tRNA.
  • CCA Can Carry Amino acids.
• T-arm
  
  • Contains the TΨC (thymine, pseudouridine, cytosine) sequence necessary for tRNA-ribosome binding.
• D-arm
  
  • Contains dihydrouracil residues necessary for tRNA recognition by the correct aminoacyl-tRNA synthetase.
• Acceptor stem
  
  • The 3′ CCA is the amino acid acceptor site.

Question 32

tRNA Charging

Answer 32

• Aminoacyl-tRNA synthetase (1 per amino acid; “matchmaker”; uses ATP) scrutinizes amino acid before and after it binds to tRNA.
  
  • If incorrect, bond is hydrolyzed.
  • The amino acid-tRNA bond has energy for formation of peptide bond.
  • A mischarged tRNA reads usual codon but inserts wrong amino acid.
• Aminoacyl-tRNA synthetase and binding of charged tRNA to the codon are responsible for accuracy of amino acid selection.

Question 33

tRNA wobble

Answer 33

• Accurate base pairing is required only in the first 2 nucleotide positions of an mRNA codon, so codons differing in the 3rd “wobble” position may code for the same tRNA/amino acid (as a result of degeneracy of genetic code).

Question 34

Protein Synthesis:
Initiation

• Initiation
• Eukaryotic vs. prokaryotic subunits
• ATP vs. GTP

Answer 34

• Initiation
  
  • Initiated by GTP hydrolysis
  • Initiation factors (eukaryotic IFs) help assemble the 40S ribosomal subunit with the initiator tRNA and are released when the mRNA and the ribosomal 60S subunit assemble with the complex.
• Eukaryotic vs. prokaryotic subunits
  
  • Eukaryotic subunits: 40S + 60S Ž–> 80S (Even).
  • PrOkaryotic subunits: 30S + 50S –> 70S (Odd).
• ATP vs. GTP
  
  • ATP—tRNA Activation (charging).
  • GTP—tRNA Gripping and Going places (translocation).

Question 35

Protein Synthesis:
Elongation & Termination

• Elongation
• Termination
• Mnemonic

Answer 35

• Elongation
  
  1. Aminoacyl-tRNA binds to A site (except for initiator methionine)
  2. rRNA (“ribozyme”) catalyzes peptide bond formation, transfers growing polypeptide to amino acid in A site
  3. Ribosome advances 3 nucleotides toward 3′ end of mRNA, moving peptidyl tRNA to P site (translocation)
• Termination
  
  • Stop codon is recognized by release factor, and completed polypeptide is released from ribosome.
• Think of “going APE”:
  
  • A site = incoming Aminoacyl-tRNA.
  • P site = accommodates growing Peptide.
  • E site = holds Empty tRNA as it Exits.

Question 36

Posttranslational Modifications

• Trimming
• Covalent alterations

Answer 36

• Trimming
  
  • Removal of N- or C-terminal propeptides from zymogen to generate mature protein (e.g., trypsinogen to trypsin).
• Covalent alterations
  
  • Phosphorylation, glycosylation, hydroxylation, methylation, acetylation, and ubiquitination.

Question 37

Chaperone protein

Answer 37

• Intracellular protein involved in facilitating and/or maintaining protein folding.
• In yeast, some are heat shock proteins (e.g., Hsp60) that are expressed at high temperatures to prevent protein denaturing/misfolding.