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Flashcards in EoM 2 Deck (90):
1

Structure of nucleotides

Nitrogenous base (purines with 2 rings: adenine,guanine; pyrimidines with 1 ring: cytosine, uracil, thymine)
5C sugar: ribose in RNA with OH at C2; 2-deoxyribose in DNA
Phosphate: high energy bond can be cleaved

2

Nucleoside

Nitrogenous Base + 5C sugar

3

Functions of nucleotides

Carry genetic info as precursors to nucleic acids (NTP, dNTP)
Carry energy (ATP, GTP)
Carry specific building blocks (UDP-glucose donates sugars in synthesis of glycogen and glycoproteins)
Regulatory signals (cAMP, 2nd messenger, cGMP) since highly charged phosphates can turn on or off signal, signal transduction cascades
Methyl donor (S-anenosyl methionine SAM)
Coenzymes (NAD, NADP, FAD, coenzyme A) carry acyl groups in metabolism, alcohol abuse affects NAD+/NADH ratio
Drugs that use nitrogenous base or nucleotide analogs (5-fluorouracil for cancer treatment and AZT anti HIV drug)

4

Primary structure of DNA

Covalent makeup of nucleotide base pair sequence, written 5'-->3' direction with A, T, C, and G, hydrophilic phosphodiester backbone with ribose sugar and phosphate on outside and hydrophobic base pairs connected by Hydrogen bonds on inside
Linear sequence of nucleotides
Anti cancer drug Actinomycin D intercalates between planar bases and interferes with DNA replication by DNA polymerase
H bonds between base pairs help keep DNA together
Strongest force keeping DNA together is interaction between planar groups of base pairs that stack like coins

5

Secondary structure of DNA

DNA double helix, stable structure taken up by some or all the nucleotides
2 strands coiled around axis of symmetry in double helix, major groove with more room for interactions with other proteins and minor groove with minimal strand separation
Usually right handed double helix (B-DNA) with outer sugar-phosphate BB that's hydrophilic and inner nitrogenous bases that are hydrophobic stacked regions
DNA must be in single stranded form to access DNA
Denaturation is disruption of non-covalent interactions holding duplex together by heat, extreme pH, or urea

6

Hyperchromic effect

Difference in absorption of 260 nm UV light between single and double stranded DNA as you increase temp, single stranded DNA has higher absorbance of 260 nm light and lower viscosity
Higher temperature promotes desaturation
Melting point in middle depends on GC and AT content
Higher GC content means it requires higher temp to denature

7

Tertiary structure

Complex folding of eukaryotic chromatin and bacterial nucleotides to make large chromosomes, double helix winds about itself and interacts with different proteins
High order tertiary structure has DNA double helix wrapped around nucleosides and nucleosides interact with each other
Dynamic structure
Tertiary structure is very important and can be passed down from generation to generation with epigenetic modifications

8

Chromatin

Complex of nucleic acids (DNA, RNA) and proteins (histones and nonhistones) comprising eukaryotic chromosomes
Lots of bases wrapped around nucleosomes, essential medium for transcriptional factors and signaling pathways altering gene activity and cell phenotype
Aberrations in it cause disease
DNA wraps around histones to form nucleosomes that form more complex/condensed chromosomes
Euchromatin is loosely packaged and transcriptionally active
Heterochromatin is tightly packaged and transcriptionally inactive

9

Histones

Have high concentration of basic amino acids like lysine and arginine to attract negatively charged DNA, histone core includes 2 copies of H2A, H2B, H3, and H4
Histone H1 associates with linker DNA between nucleosides to help package chromatin to higher order structures

10

Nucleosomes

DNA strand wrapped around histone core with 2 copies of H2A, H2B, H3, and H4

11

Differences in packaging of DNA in viruses, bacteria, and higher organisms

Viruses has genome surrounded by protein coat
9,000 nucleotides
Bacteria have circular chromosome and smaller circular DNAs called plasmids that can give antibiotic resistance
4.5 million base pairs
Human genome has 3 billion base pairs and 2 copies, eukaryotic DNA packaged into chromatin so it can be accessed
Huge differences in amount of DNA that can be replicated and accessed in single stranded form, so it must be packaged efficiently

12

Eukaryotic chromosomal structure

The chromosome is a linear structure in humans, organized into chromosomes in vivo (circular in mitochondria and bacteria)
Chromosome has:
-centromere for spindle fiber attachment in mitosis and meiosis, joins 2 sister chromatids
-Telomere repeated DNA sequence at end of eukaryotic chromosomes to protect them during cell division, provides mitotic clock since 100 nucleotides are lost from ends during each division
-Origins of replication (nucleotide sequence where DNA synthesis begins called ori site), we have 1 every 50,000 base pairs while DNA have just 1 in their circular genome
We have 23 pairs of chromosomes in our karyotype

13

Epigenetics

Regulate gene expression through chromatin conformation
Crucial during development, disease states, and cellular differentiation
Change gene expression by environment, including diet
Queen bee morphology and life span due to epigenetic changes that occur in bee when fed royal jelly as larvae
Changes tertiary structure, same genetic sequences
Chromatin structure affects DNA expression

Methylation typically occurs at cytosine residues with CPG dinucleotide
Can sterically inhibit binding of trans-action factors, typically repressing transcription if it is at promoter region
Can bind repressive factors and activate transcription
Serves as recognition motif for binding specific factors
adds methyl to non H-bonding site, so it doesn't change information carrying capacity but forms motif recognizable by specific binding proteins and can sterically inhibit interactions with DNA molecule while serving as binding site for proteins
Methyl CPG binding protein binds to motif and causes Rhett syndrome by forming place for other proteins to interact and inhibit things from interacting; DNA methyltransferase adds methyl group to cytosine using SAM
Epigenetic modifications can be passed down to offspring

Histone modifications (many in list) including acetylation that adds acetyl to amino group of lysine and partially neutralizes positively charged histone (histone has lots of lysines) and decreases the histone affinity for negatively charged DNA
Prevents formation of tight histone and prevents higher structure by loosening chromatin
Makes chromatin more active in decondensed form
Nucleosomes can't interact as well
Epigenetic modification can include post translational modification to amino terminal tail and internal sites of histones

14

Acyclovir

Discovered by Elion and Hitchings
Nucleotide analog that's a chain terminator, targets viral DNA polymerases of chicken pox and herpes virus
Phosphorylated by viral thymidine kinase that ensures specificity
Structure: guanine attached to incomplete ribose ring lacking 3' OH needed for polymeration, so it acts as chain terminator
Esterification increases its bioavailability

15

3 rules of DNA replication for prokaryotes and eukaryotes

DNA replication is semi-conservative: start with double helix and each parental strand is used as template to make new strand; each daughter duplex consists of 1 parental and 1 newly synthesized strand

Replication begins at origin and proceeds bidirectionally, origins of replication are AT rich sites with weaker bonds to facilitate chain separation; moves in both directions away from origin and forms replication bubble and forks eventually meet up and get terminated to get 2 new chromosomes (2 strands separate and form V shaped fork structure where DNA is made); There are 4 sites of DNA replication

DNA synthesis I goes in 5'-->3' direction and is semidiscontinuous
Discontinuous on lagging strand with Okazaki fragments; continuous on leading strand; deoxynucleotides are added to 3' end; RNA primer must be laid down to initiate polymerase activity; new strand is complementary to parent template

16

DNA replication ingredients

Triple A ATPase (DnaA) uses energy of ATP cleavage to separate 2 strands
DNA ligament seals nicks in DNA (breaks in sugar-phosphate backbone) with phosphodiester bonds
DNA polymerase require RNA primer and ssDNA as template to elongate strand and proofread
Helicase dissociates 2 strands and pushes replication forward
Endonuclease (within) and exonuclease (at ends) put nicks in DNA backbone by severing phosphodiester bonds
Primase provides primer for DNA polymerase, makes short stretches of RNA complementary to DNA strand
Topoisomerase II (gyrase) alleviates positive supercoiling stress in front of fork, it's an endonuclease and ligase, can cut strands and allow them to unwind to reduce supercoiling stress and reseals the strands in eukaryotic and prokaryotic DNA replication

17

Steps of DNA replication in prokaryotes

Initiation at origin of replication:
-DnaA binds origin of replication at specific AT rich sequence
-Helicase disassociates double helix (it binds to fork in initiation and pushes fork forward in elongation)
-single strand DNA binding protein protects DNA and maintains disassociation to prevent it from reannealing

Elongation at both forks:
-RNA primase to lay down RNA primers so
-DNA polymerase III can start DNA synthesis from 3' end of primer; synthesized in 5' to 3' direction on leading and lagging strands (discontinuous with Okazaki fragments); new strands are complementary and antiparallel to parental strand; proofreading functions are present to spot if wrong base is incorporated (tautomeration can cause this), purines-pyrimidine pair fits in active site, nucleotides are added one at a time in 5'-3' direction, and 3'-5' exonucleases cleave off wrong nucleotide so DNA poly 3 can proceed in 5'-3' direction;
-Topoisomerases use energy of ATPase to alleviate supercoiling stress in front of fork by unwinding and resealing strands and breaks apart catenated chromosomes (2 rings hooked together);
-On lagging strand, DNA poly 1 removes RNA primers with 5'-3' exonuclease and replaces them with DNA starting at 3' end of fragments; uses dNTP and dNMP and 2 phosphates to drive synthesis;
-DNA ligase seals nicks
Topoisomerase is targeted by drugs to interfere with catalytic cycle, topoisomerase II poisons stabilize the topoisomerase complex for antitumor activities, while catalytic inhibitors act on other steps in catalytic cycle

Termination: when 2 replication forks meet up; proteins resolve the 2 newly formed chromosomes and termination sequences stop fork from proceeding; you can be left with catenated chromosomes that topoisomerase II gyrase resolves (topoisomerase is in elongation and termination phase)


18

Roles of DNA polymerases in prokaryotes

DNA poly 1: DNA repair and removes RNA primers during replication with 5'-3' exonuclease activity, only does 10-20 nucleotides/second to add short stretches after it deletes RNA primers

DNA poly II, IV, and V: DNA repair

DNA poly III: the workhorse for prokaryotic DNA replication, 1,000 nucleotides/second, has extra protein structures to latch onto DNA

DNA poly 1 and 3 have proofreading functions (aspect of 3'-5' exonuclease activity)

19

Concept of fidelity of genetic information and how it relates to DNA replication

Fidelity=accurately passing on genetic code during DNA replication

-Less than 1 mutation per cell division in humans due to proofreading
-geometry of active site of DNA polymerase improves fidelity, has purine-pyrimidine hydrogen bonding
-proofreading is done with 3'-5' exonuclease activity
-Mismatch repair with DNA methylation as a defense mechanism so it can recognize it's own DNA, newly synthesized strands in error aren't methylated and can therefore be differentiated from old methylated strands and cleaved out

20

Etiology and symptoms of mitochondrial DNA depletion disorders and how they affect more than 1 organ

Often involves multiple systems
Mitochondrial DNA depletion disorders result from defects in Mt genomes with defective gamma DNA polymerase or deletion of Mt DNA (2-10 copies of its genome are present)
Mt DNA depletion or dysfunction of DNA polymerase gamma is the cause
Has cytoplasmic segregation with random distribution of Mt genome in mitosis/meiosis instead of using mitotic spindle to segregate equal amounts of DNA to separate daughter cells in nucleus

Functionality is encoded in nuclear genome (proteins for Mt genome are encoded in nuclear genome and shuttled to Mt genome
The Mt genome doesn't get mutated. The replicating factor that copies the Mt genome gets mutated
Mutations occur in nuclear genes that are transported to Mt
Inherited in autosomal recessive fashion

Etiology isn't well known, affects neuromuscular, ophthalmologist, and GI systems, especially liver failure
3+ organ systems involved strongly indicates Mt disease

There is a link between strong effect size (mutations with strong dysfunction causing multi-system disorders) and heteroplasmy
Mutations causing severe disorders are rare because those who get them don't live past childhood and can't pass them on to future generations

Polymorphisms causing little dysfunction in homoplastic state are more common and liked to disease such as Parkinson's disease
Weak dysfunction alleles are typically just linked with disease, causing increased susceptibility rather than causing the syndrome itself

21

MT DNA depletion syndromes

Progressive disease affecting children, those treated for HIV and Hep B (nucleotide analog targets replication machinery of HIV but also inhibits functioning of gamma DNA polymerase, causing Mt DNA depletion syndromes), and young adults
Mt disorders related to DNA polymerase gamma dysfunction are most common
Defects in replication of Mt DNA with DNA polymerase gamma results in deletion of DNA and multi system disturbance, especially in high energy organs like the liver and brain
13 members of ETC are encoded in Mt DNA and loss inhibits oxidative phosphorylation
Accumulation of Krebs cycle intermediates is also evidence of Mt dysfunction
Elevated tyrosine associated with Alpers syndrome
Diseases linked to Mt DNA depletion: Alpers Syndrome with liver failure, epilepsy, and retardation; progressive external ophthalmoplegia affecting ocular motility, and Ataxia-neuropathy syndrome with loss of kinesthetic and vibratory sensation and reflexes
Aging is associated with Mt DNA damage too

22

Endosymbiotic Theory

Mt originated from aerobic free living bacteria that were engulfed by eukaryotic cell that evolved in symbiosis with host cell

23

Heteroplasmy affects Mt DNA depletion syndromes

Heteroplasmic since each cell contains different types of Mt DNA at varying levels, causing a complicated disease pattern when deleterious mutations are present (separates Mt. genome into different organelles) 1 cell contains multiple copies of Mt DNA randomly distributed, and wild and mutant Mt DNA can coexist in heteroplasmy

24

Replication of linear chromosomes in eukaryotic DNA

Many different origins of replication in eukaryotes (instead of 1 as in prokaryotes), spaced every 30,000-300,000 base pairs since it has lots more DNA to replicate and is slower process due to complex tertiary chromatin structure
Initiate replications at both sides of fork bidirectionally with same rules mentioned before to get 2 linear chromosomes at end of replication
Oris are just any stretch of AT rich DNA in eukaryotes that enzymes can latch onto to replicate
Poly alpha: contains primase, lays down RNA primer to start DNA synthesis
Poly beta: repairs
Poly gamma: replicates Mt DNA
Poly delta: elongates Okazaki fragments of lagging the strand
Poly epsilon: elongates leading strand
Delta and Gamma are the workhorses in eukaryotic DNA replication
Gamma, delta, and epsilon have 3'-5' exonuclease proofreading functions
Cell cycle involves G1, S-phase with replication, G2, and mitosis with separation of 2 cells and checkpoints along the way to ensure fidelity of the information.
Cancer results if checkpoints fail)
Eukaryote DNA replication is more complex and regulated, 50 bps/second instead of 1,000 bps/second due to differences in tertiary structure (1/20th the speed)--DNA must be disassociated from chromatin and converted to single stranded form before it can be accessed and replicated
More origins of replication with nonspecific AT rich sequences
Telomere replication differs
Greek symbols instead of Roman numerals (prokaryotic)
Both prokaryotic and eukaryotic DNA replication is semi conservative

25

Problems with linear chromosomes: telomerase

Telomeres protect ends of linear chromosomes to prevent chromosomal fusion (distinguish ends from double stranded DNA breaks) and allow proper functionality
Full replication of ends is important in cellular senescence when it enters G0 and won't divide because telomeres are too short and in cancer when it enters cellular crisis and survives and becomes malignant
Telomerase (reverse transcriptase~RNA dependent DNA polymerase) produces repetitive sequences at ends of chromosomes
Telomerase is in stem cells to allow them to divide as many times as they want because telomeres keep regenerating
Telomerase also immortalizes cancer cells so they start dividing again
Telomeres are important at ends of linear chromosomes (not circular bacterial chromosomes)
Has unique telomere sequence at centromere, then buffer region of telomere associated repeats, then the telomeretric sequence (TTaGGG repeats), then single stranded region folded upon itself and bound by proteins

26

Telomerase mechanism

Telomerase is an RNA dependent DNA polymerase that encodes a sequence to extend 3' end of single stranded DNA by reverse transcription to get 3' overhang

The newly synthesized strand with RNA primer removed has 3' overhang
Telomerase extends 3' end of DNA further to provide a template
Primase provides RNA primer
DNA polymerase extends RNA primer on non-overhanging strand
RNA primer is removed to result in preservation or extension of telomeres 5bp repeat, leaving 3' overhang

27

Different types of eukaryotic RNA and their roles in gene product generation

RNA polymerase I makes precursor of rRNAs (18S, 28S, 5.8S)

RNA poly II transcribes all mRNA genes that will be translated to protein, miRNAs, snRNA for splicing
-all poly II transcripts are polyadenylated

RNA poly III transcribes tRNAs, U6 snRNA, and 5S rRNA

They share 9 core subunits and require general transcription factors to start transcription

28

Housekeeping genes

In most cell types for cell structure and metabolism
Constitutive=expressed in same amount in all tissues

29

Tissue specific genes

Cell type specific and highly regulated
-globin in only RBCs

30

Non-standard bases in RNA impact RNA structure

Eukaryotic promoters: BRE element at -35 to bind transcriptional complex
TATAA box at -25 to -30 to bind general TFs
Inr initiator elements at transcription start sites to allow RNA polymerase to bind
Downstream elements (DPE, DCE, MTE) to bind other TFs

These elements guide transcription complex to promoter region

31

Steps of eukaryotic transcription initiation
There is a stepwise model described below and a holoenzyme model in which RNA polymerase II forms complex with general TFs that's recruited to promoter by activation factors

TBP associated factors (TAFs)

TFIID recognizes TATAA box at promoter and drags TFs (TBP and TAFs) with it
Kinks double stranded molecule so TFIIB can bind to BRE element adjacent to TATAA
Now TFIIF and RNA polymerase II can bind
The TBP-TFIID complex binds to TFIIF-RNA poly II complex
TFIIE and TFIIH bind to complete preinitiation complex
TFIIH has helicase to unwind double helix
Mediator contacts general TFs and RNA poly II to increase transcriptional activity and mediate signal until CTD is phosphorylated and can bind other proteins to facilitate elongation
Using ATP, TFIIH phosphorylates serine residues in carboxy terminal domain (CTD) which adds negative charges and disrupts and dissociates Poly 2 from preinitiation complex
Transcription is initiated

32

Eukaryotic cells have complex chromatin structure as regulatory model

Chromatin allows DNA to be condensed or non condensed
HAT acetylates, decondenses, and activates chromatin and removes positive charge from lysine
HDAC condenses and inactivates chromatin (deacetylated form of histone)

33

The conversion of precursor rRNA to mature rRNA happens in nucleolus
Involves 2 sets of cleavages
Linkage through H-bonding
Methylation of bases and sugar residues by snoRNA and snoRNP
Chemical conversion of bases (uridine to pseudouridine)

RNA polymerase 1 transcribes rRNA genes
Primary step with endonuclease gives 45S transcript Secondary step with RNases (endonuclease) yields 28S, 18S, 5.8S rRNAs
Endonucleases further cleave unneeded portions to get mature rRNAs with 5.8S rRNA H-bonded to 28S rRNA

SnoRNA and snoRNPs participate in cleavage and base modifications (add methyl groups to bases within rRNAs and convert uridine residues to pseudouridines)

34

RNA polymerase III transcribes tRNA, U6 snRNA, and 5S rRNA

RNA polymerase III transcribes tRNA and 5S rRNA with transcription factors downstream of transcription start site
RNA poly II also transcribes U6 snRNA
U6 snRNA, tRNA, and 5S rRNA require specialized combinations of TFs to initiate transcription by recruiting RNA poly III

35

TRNA processing with cleavage of 3' and 5' ends, attachment of CCA bases for AA attachment, and replacement of standard RNA bases at 10% of its positions

Starts with pre-tRNA
RNAse P has catalytic RNA component (ribozyme that is active without protein component) to cleave 5' end of pre-tRNA
Another RNAse cleaves 3' end of tRNA, often processed with addition of CCA end for amino acid attachment
10% of tRNA bases are modified to change base pairing properties and stabilize hairpin loops
Involves lots of chemical modifications to get nucleotides with different properties

36

In prokaryotes, transcription is coupled with translation
In eukaryotes, mRNA after processing (capping 5' end with 7-methylguanosine; tailing 3' end with polyadenylation; splicing with removal of introns and joining exons) is moved to cytosol for translation
Process of mRNA capping:

7-methylguanosine Cap is added to 5' end with 5' to 5' triphosphate linkage
A methylated GTP is added to 5' end by 7-methylguanisine cap
The ribose of first 2 bases is methylated too

The cap stabilizes mRNA so it can align with ribosome

37

Processing eukaryotic mRNA
Polyadenylation:

Endonuclease cleaves precursor at CA to generate 3' end
Poly-A-polymerase adds poly A tail of 200 bases to 3' end of pre-mRNA during processing
The enzymes are attracted to phosphorylated CTD of RNA polymerase

38

Processing of mRNA
Splicing occurs in spliceosomes
5 snRNAs (there are 6 known) complex with 6-10 proteins to form snRNPs that position reactive bases
Some mRNAs are self splicing with catalytic RNA residues for folding
Most mRNAs require snRNP complexes to fold mRNA so that reactive molecules are close together
Conserved sequences at 5' and 3' end for binding

U1snRNP binds 5' splice site (GU)
U2 contacts conserved branch point A and U1 to bend mRNA and bring exon 1 closer to exon 2
U4/U6 and U5 complex enters spliceosome
U5 holds the first exon (binds upstream of 5' splice site)
U1 and U4 leave
U6 complexes with U2 and conformational change occurs with cleavage of exon 1 from the intron at 5' splice site
5' end of intron forms phosphodiester bond with internal adenine to form lariat
U5 binds 3' splice site and leads to cleavage, removal of lariat, and formation of phosphodiester bond between 3' OH of exon 1 and 5' phosphate of exon 2

Introns are removed and exons are linked together

Cleavage between exon 1 (GU sequence) and intron sequence at 5' end leaving an exon with free 3' end
Linkage between first base in intron and catalytic Adenine residue in branch point to form phosphodiester linkage forming lariat
2nd cleavage at 3' end separates exon 2 (AG sequence) from lariat shaped intron
Transesterification joins 3' end of exon 1 with 5' end of exon 2

39

Controlling mechanisms of splicing so that it is sequential
Joins spliceosome so that it only reacts with correct intronic sequences
Maintain correct order of splicing through coupling it to transcription
Mediate alternative splicing to make several different mRNAs and protein products
1 mRNA can yield different protein products with different functions for increased diversity
Removing exotic sequences changes function of protein, may stimulate or repress ability of snRNPs to bind

SR proteins recruit snRNPs for spliceosome assembly
U2AF binds 3' splice sites and recruits U2 to branch point

40

RNA editing: changing bases to alter amino acid sequence of protein product

RNA editing changes coding capacity of mRNA so different protein products can be made
Edited RNA can create stop codon
Unedited RNA may encode a longer protein

41

RNA degradation

Over 90% of pre mRNA is introns and must be degraded
RNAs are turned over
Amount of mRNA is determined by amount of production versus loss due to degradation/turnover
Half life is determined by length of poly A tail
3' and 5' naked ends and lariat with 2'-5' phosphodiester bond are targeted
Standard mRNA is protected at both ends by 5' 7-methylguanosine cap and 3' poly A tail
Exonuclease will attack if poly A tail or 7-methylguanosine is removed

42

Branch point at A residue is highly conserved

Branch point=UAC UAAC

43

Central Dogma

Replication of DNA
Transcription: DNA--RNA
Translation: RNA--protein

44

Reverse transcriptase

Viruses take RNA and reverse transcribe to DNA using reverse transcriptase
We study gene expression this way (PCR: isolate RNA and reverse transcribe to cDNA and read level of gene expression)

45

DNA repair, mutation, recombination (rearranges genetic material), processing of RNA and protein, nucleotide metabolism of DNA and RNA

All processes can be regulated
Mutations (mistakes) are the raw materials for evolution

46

Types of pathways

De novo biosynthesis creates new molecules
Salvage biosynthesis sales bits and pieces
Degradation breaks down products

47

De novo biosynthesis of purines starts with pentode sugar and slowly builds purine ring

De novo synthesis of pyrimidines builds pyrimidine ring then attaches sugar at end

48

De novo Purine biosynthesis
2 regulated steps, 2nd step is committed
2, 5, 15 use glutamine
4, 10 use folate
Ribose-5-phosphate---PRPP---5-phosphoribosylamine---IMP splits to form either adenylosuccinate, AMP, ADP, and ATP or
XMP, GMP, GDP, and GTP
2 sides of pathway (ATP and GTP) can boost each other's production and balance each other

1st regulated step: ribose-5-phosphate---PRPP synthetase adds phosphate---PRPP
Negative feedback by purine nucleotides
2nd regulated and committed step: PRPP---PRPP glutamyl amidotransferase with glutamine---5-phosphoribosylamine
Negative feedback by purine nucleotides and activated by PRPP
Form IMP with base hypoxanthine (know where C and Ns come from)
Splits at IMP to get to usable adenosine monophosphate or guanosine monophosphate (AMP or GMP)

49

Inhibitors of Purine biosynthesis

Glutamine analog Azaserine (blocks steps 2, 5, 15)
Purine inhibitors target cancer that's rapidly dividing (plus hair and intestinal cells)
Folate metabolism (block steps 4 and 10) inhibitor Sulfonamide (inhibits bacterial production of folic acid from PABA)
Folate metabolism inhibitor methotrexate for cancer treatment
-Bacteria convert PABA to folic acid; we convert folic acid to tetrahydrofolate using dihydrofolate reductase
Tetrahydrofolate (THF) donates a carbon to build ring and makes folate derivatives necessary for purine synthesis (steps 4 and 10)
Methotrexate and Sulfonamides inhibit cofactor folic acid used in purine synthesis
Low folic acid causes spina bifida and anencephaly due to low purine synthesis

50

Purine salvage

Phosphorylate purine nucleosides (adenosine) to AMP
Phosphoribosylate purine base (adenine) with transferase and PRPP to form AMP
Phosphoribosylate hypoxanthine--IMP or guanine--GMP with HGPRT
HGPRT is most important enzyme
Low HGPRT allows PRPP accumulation with more purine synthesis and build up of Uric acid as final product of degradation pathway and causes Lesch-Nyhan Syndrome

51

Lesch-Nyoman syndrome

Deficiency of HGPRT with PRPP accumulation that activates purine synthesis which means more purines must be degraded and final product in degradation pathway is uric acid
The syndrome is x-linked recessive, purine overproduction, gouge and kidney stones, neurological problems and retardation, bizarre self-mutilation

52

Purine degradation

Excess of purines
Adenosine or guanosine loses amino group--inosine
Purine nucleotide phosphorylase removes sugar--hypoxanthine or guanine
Guanine and hypoxanthine are converted to xanthine
Xanthine converted to uric acid that accumulates in joints and kidneys

53

Purine degradation
ADA deficiency

Normally converts adenosine to inosine
Adenosine deaminase (ADA) deficiency causes SCID with no T or B-cells

54

Purine degradation
Purine nucleoside phosphorylase deficiency

Normally removes ribose sugar to convert inosine to hypoxanthine or guanosine to guanine
Purine nucleoside phosphorylase deficiency causes autosomal recessive disease with immunodeficiency and no T-cell production

55

Purine degradation
Allopurinol xanthine analog

Allopurinol blocks conversion of hypoxanthine to xanthine to uric acid
Allopurinol blocks conversion of xanthine to uric acid to treat gout and prevent formation of uric acid crystals

56

Pyrimidine de novo biosynthesis starts with pyrimidine ring then attaches sugar at end

C02+glutamine+ATP---carbamoyl phosphate synthetase II in cytoplasm---CAP
Stimulated by ATP from purine synthesis
Inhibited by UTP pyrimidine synthesis end product
Orotic acid is first compound with pyrimidine ring
Orotic acid--OMP---orotidylic acid decarboxylase---UMP---rotate phosphoribosylate transferase---UDP
Splits at UDP to get CTP or TMP

57

Orotic aciduria associated with pyrimidine de novo biosynthesis

Due to deficiency in orotidylic acid decarboxylase and/or orotate phosphoribosylate transferase

58

Folate deficiency (folate used in last step to make TMP in de novo pyrimidine synthesis) means thymidylate synthase can't make TMP

Fluorine attached to dUMP means thymidlate synthetase and methylene THF stay attached (going from dUMP--TMP), causing suicide inhibition

59

Pyrimidine salvage

Salvage pyrimidine nucleosides
Uridine or cytidine---ATP---UMP or CMP
Thymidine---ATP---TMP

60

Thymidine kinase is used to salvage thymidine---TMP
Target thymidine kinase with Acyclovir (guanosine analog) for viral therapy

The guanosine analog Acyclovir is recognized by viral thymidine kinase
Acyclovir blocks thymidine kinase in bacteria to decrease viral DNA synthesis and reproduction

61

HAT Growth medium
Hypoxanthine, Aminopterin, thymidine

Normal cells can live and grow, but Lesch-Nyhan cells die
Lesch-Nyan cells lack HGPRT and can't salvage hypoxanthine or guanine for pyrimidine synthesis
Aminopterin is like methotrexate and inhibits folate metabolism required for de novo purine (4 and 10) and pyrimidine synthesis (last step to make TMP)
Must use thymidine salvage pathway of pyrimidine synthesis with thymidine kinase to make TMP
To live and grow, cells must have HGPRT and thymidine kinase

62

Form deoxynucleotides for DNA with ribonucleotide reductase that gets reducing power from thioredoxin

NDP (C, U, A, or G)---ribonucleotide reductase with thioredoxin for reducing power--dNDP
NDP (T) comes from adding methyl group to dUMP during pyrimidine de novo synthesis

63

PRPP synthetase leads to overproduction of purines

HGPRT deficiency leads to Lesch-Nyhan syndrome
And it causes excess PRPP that further increases purine production

64

Adenosine deaminase deficiency

Causes SCID with no T or B cells
Adenosine deaminase required in purine degradation for converting adenosine to inosine

65

Purine nucleoside phosphorylase deficiency

Causes no T cell production
Used to convert inosine to hypoxanthine and guanosine to guanine by removing sugar
Ribose-5-phosphate---PRPP synthetase---PRPP---PRPP glutamyl amidotransferase---5-phosphoribosylamine
IMP with split to form GTP or ATP
Degradation: Adenosine---adenosine deaminase---inosine---purine nucleotide phosphorylase removes sugar---hypoxanthine--xanthine---uric acid that can accumulate
Guanosine---purine nucleotide phosphorylase---guanine---xanthine---uric acid that can accumulate

66

Metabolic disorders in pyrimidine synthesis
Orotic acidurias

Type 1 involves deficiency of Orotate phosphoribosyltransferase and orotidylate decarboxylase
Give uridine so cells can convert it to UMP (salvage of pyrimidines) which goes on to form CTP and TMP
CO2+glutamine+ATP--CAP--orotic acid with pyrimidine ring---OMP and UMP (orotate enzymes)---UDP with split to form CTP or TMP

67

Central Dogma

Replication of DNA
Transcription: DNA--RNA
Translation: RNA--protein

68

Reverse transcriptase

Viruses take RNA and reverse transcribe to DNA using reverse transcriptase
We study gene expression this way (PCR: isolate RNA and reverse transcribe to cDNA and read level of gene expression)

69

DNA repair, mutation, recombination (rearranges genetic material), processing of RNA and protein, nucleotide metabolism of DNA and RNA

All processes can be regulated
Mutations (mistakes) are the raw materials for evolution

70

Types of pathways

De novo biosynthesis creates new molecules
Salvage biosynthesis sales bits and pieces
Degradation breaks down products

71

De novo biosynthesis of purines starts with pentode sugar and slowly builds purine ring

De novo synthesis of pyrimidines builds pyrimidine ring then attaches sugar at end

72

De novo Purine biosynthesis
2 regulated steps, 2nd step is committed
2, 5, 15 use glutamine
4, 10 use folate
Ribose-5-phosphate---PRPP---5-phosphoribosylamine---IMP splits to form either adenylosuccinate, AMP, ADP, and ATP or
XMP, GMP, GDP, and GTP
2 sides of pathway (ATP and GTP) can boost each other's production and balance each other

1st regulated step: ribose-5-phosphate---PRPP synthetase adds phosphate---PRPP
Negative feedback by purine nucleotides
2nd regulated and committed step: PRPP---PRPP glutamyl amidotransferase with glutamine---5-phosphoribosylamine
Negative feedback by purine nucleotides and activated by PRPP
Form IMP with base hypoxanthine (know where C and Ns come from)
Splits at IMP to get to usable adenosine monophosphate or guanosine monophosphate (AMP or GMP)

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Inhibitors of Purine biosynthesis

Glutamine analog Azaserine (blocks steps 2, 5, 15)
Purine inhibitors target cancer that's rapidly dividing (plus hair and intestinal cells)
Folate metabolism (block steps 4 and 10) inhibitor Sulfonamide (inhibits bacterial production of folic acid from PABA)
Folate metabolism inhibitor methotrexate for cancer treatment
-Bacteria convert PABA to folic acid; we convert folic acid to tetrahydrofolate using dihydrofolate reductase
Tetrahydrofolate (THF) donates a carbon to build ring and makes folate derivatives necessary for purine synthesis (steps 4 and 10)
Methotrexate and Sulfonamides inhibit cofactor folic acid used in purine synthesis
Low folic acid causes spina bifida and anencephaly due to low purine synthesis

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Purine salvage

Phosphorylate purine nucleosides (adenosine) to AMP
Phosphoribosylate purine base (adenine) with transferase and PRPP to form AMP
Phosphoribosylate hypoxanthine--IMP or guanine--GMP with HGPRT
HGPRT is most important enzyme
Low HGPRT allows PRPP accumulation with more purine synthesis and build up of Uric acid as final product of degradation pathway and causes Lesch-Nyhan Syndrome

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Lesch-Nyoman syndrome

Deficiency of HGPRT with PRPP accumulation that activates purine synthesis which means more purines must be degraded and final product in degradation pathway is uric acid
The syndrome is x-linked recessive, purine overproduction, gout and kidney stones, cerebral palsy with neurological problems and mental retardation, bizarre self-mutilation

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Purine degradation

Excess of purines
Adenosine or guanosine loses amino group--inosine
Purine nucleotide phosphorylase removes sugar--hypoxanthine or guanine
Guanine and hypoxanthine are converted to xanthine
Xanthine converted to uric acid that accumulates in joints and kidneys

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Purine degradation
ADA deficiency

Normally converts adenosine to inosine
Adenosine deaminase (ADA) deficiency causes SCID with no T or B-cells

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Purine degradation
Purine nucleoside phosphorylase deficiency

Normally removes ribose sugar to convert inosine to hypoxanthine or guanosine to guanine
Purine nucleoside phosphorylase deficiency causes autosomal recessive disease with immunodeficiency and no T-cell production

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Purine degradation
Allopurinol xanthine analog

Allopurinol blocks conversion of hypoxanthine to xanthine to uric acid
Allopurinol blocks conversion of xanthine to uric acid to treat gout and prevent formation of uric acid crystals

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Pyrimidine de novo biosynthesis starts with pyrimidine ring then attaches sugar at end

C02+glutamine+ATP---carbamoyl phosphate synthetase II in cytoplasm---CAP
Stimulated by ATP from purine synthesis
Inhibited by UTP pyrimidine synthesis end product
Orotic acid is first compound with pyrimidine ring
Orotic acid--OMP---orotidylic acid decarboxylase---UMP---rotate phosphoribosylate transferase---UDP
Splits at UDP to get CTP or TMP

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Orotic aciduria associated with pyrimidine de novo biosynthesis

Due to deficiency in orotidylic acid decarboxylase and/or orotate phosphoribosylate transferase

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Folate deficiency (folate used in last step to make TMP in de novo pyrimidine synthesis) means thymidylate synthase can't make TMP
5-fluorouracil derivates inhibit thymidylate synthase

Fluorine attached to dUMP (F-dUMP) means thymidlate synthetase and methylene THF stay attached (going from dUMP--TMP), causing suicide inhibition

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Pyrimidine salvage

Salvage pyrimidine nucleosides
Uridine or cytidine---ATP---UMP or CMP
Thymidine---ATP---TMP

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Thymidine kinase is used to salvage thymidine---TMP
Target thymidine kinase with Acyclovir (guanosine analog) for viral therapy

The guanosine analog Acyclovir is recognized by viral thymidine kinase
Acyclovir blocks thymidine kinase in bacteria to decrease viral DNA synthesis and reproduction

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HAT Growth medium
Hypoxanthine, Aminopterin, thymidine

Normal cells can live and grow, but Lesch-Nyhan cells die
Lesch-Nyan cells lack HGPRT and can't salvage hypoxanthine or guanine for purine synthesis
Aminopterin is like methotrexate and inhibits folate metabolism required for de novo purine (4 and 10) and pyrimidine synthesis (last step to make TMP)
Must use thymidine salvage pathway of pyrimidine synthesis with thymidine kinase to make TMP
To live and grow, cells must have HGPRT and thymidine kinase

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Form deoxynucleotides for DNA with ribonucleotide reductase that gets reducing power from thioredoxin

NDP (C, U, A, or G)---ribonucleotide reductase with thioredoxin for reducing power--dNDP
NDP (T) comes from adding methyl group to dUMP during pyrimidine de novo synthesis

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Excessive PRPP synthetase leads to overproduction of purines and gout
HGPRT partial deficiency also leads to gout (backs up PRPP)

HGPRT complete deficiency leads to Lesch-Nyhan syndrome with gout and kidney stones, mental retardation, neurological problems, and bizarre mutilation
And this deficiency causes excess PRPP that further increases purine production and therefore more purines must be degraded, leading to more uric acid deposits~gout

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Adenosine deaminase deficiency

Causes SCID (severe combined immunodeficiency disease) with no T or B cells
Adenosine deaminase required in purine degradation for converting adenosine to inosine

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Purine nucleoside phosphorylase deficiency

Causes immunodeficiency with no T cell production
Used to convert inosine to hypoxanthine and guanosine to guanine by removing sugar
Ribose-5-phosphate---PRPP synthetase---PRPP---PRPP glutamyl amidotransferase---5-phosphoribosylamine
IMP with split to form GTP or ATP
Degradation: Adenosine---adenosine deaminase---inosine---purine nucleotide phosphorylase removes sugar---hypoxanthine--xanthine---uric acid that can accumulate
Guanosine---purine nucleotide phosphorylase---guanine---xanthine---uric acid that can accumulate

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Metabolic disorders in pyrimidine synthesis
Orotic acidurias

Type 1 involves deficiency of Orotate phosphoribosyltransferase and orotidylate decarboxylase
Give uridine so cells can convert it to UMP (salvage of pyrimidines) which goes on to form CTP and TMP
CO2+glutamine+ATP--CAP--orotic acid with pyrimidine ring---OMP and UMP (orotate enzymes)---UDP with split to form CTP or TMP