5.1-5.4 - DNA Replication, Transcription, Gene & Mutation

1) constitutive = ALWAYS tightly compacted (transcriptionally inactive, contains no genes, highly repetitive DNA sequences, replicated late in interphase) 2) facultative - heterochromatin that contains inactive genes (differs based on cell type and regulated based on what that cell wants to replicate)

constitutive vs. facultative heterochromatin (condensed)?

1) maximum compaction occurs during metaphase 2) DNA condensed 10,00 fold to 1.4 meters 3) most DNA is in 30nm or 10 nm (euchromatin, potential for transcription) during interphase

describe interphase DNA organization and when this would and would not likely occur

1) charged aminoacyl-tRNA binds to A site (has eEF-1a elongation factor with GTP) 2) GTP hydrolysis if correct tRNA is in place, pushing eEF1a out with GDP (wrong tRNA would normally leave before hydrolysis) 3) 23S rRNA makes bond between P and A site 4) tRNA-P site bond is broken 5) eEF-2 translocates growing uncharged tRNA with more GTP hydrolysis 6) binding of new charged tRNA triggers release of uncharged tRNA

describe the process of elongation during translation

1) eukaryotic initiation factors (eIFs) identify 5' cap and internal ribosome entry site (IRES, most often at 5' UTR) 2) eIFs assemble 40S, move in 5'-3' direction, and are released when mRNA and 60S assemble with complex [requires GTP]

describe the process of initiation of translation

1) U1 = binds at 5' splice site of pre-mRNA 2) U2 = binds branch point and interacts with U1 3) U4/U6/U5 = trimer binds (U5 binds exons at 5' site, U6 binds to U2) 4) U4 leaves with U1 5) snRNAs (not snRNPs) carry out splicing

describe the process of splicing of pre-RNA (specifically what U1, U2, U4, U5, and U6 snRNAs are responsible for)

release factors recognize stop codon and halt translation = hydrolyze GTP to release polypeptide

describe the process of termination

1) sigma subunit identifies promoter region nonspecifically (scans in either direction) 2) initially closed promoter = open promoter after 3) continues in 5'-3' direction

describe the process of transcription in prokaryotes

1) pyrimidine - cytosine and thymine 2) purine - adenine and guanine a) base linked to 1' carbon, phosphate to 5' and 3' b) 5' end always ends with phosphate, and 3' always ends with hydroxyl

differentiate between four nucleotide bases

1) general = involved in transcription from all pol II promoters 2) gene-specific - involved in expression of specific genes

differentiate between general (GTFs) and gene-specific transcription factors

1) lack of dystrophin (DMD) = progressive myofiber damage (muscle replaced with fat & connective tissue) 2) 1:3500 males, x-linked recessive (mostly nonsense mutations) 3) potential therapy with CRISPR

duchenne muscular dystrophy: inheritance, mutated protein, therapy

1) exons are coding segments, and introns are non-coding segments (usually more introns than exons in genes) 2) not all exons are always utilized (exon shuffling, alternative splicing)

exons vs. introns; are all exons utilized?

a-amanitin

found in Amanita phalloides (death cap mushrooms); inhibits RNA polymerase II in eukaryotes but can lead to severe hepatotoxicity

1) AR (very rare) 2) BLM gene - mutated helicase 3) short stature, sensitive skin, predisposition for cancer and genomic instability

gene, protein, mode of inheritance, clinical presentation for Bloom syndrome

1) x-linked (also AD and AR forms) 2) mutation in telomere (DKC1 for x-linked) 3) skin pigmentation, bone marrow failure

gene, protein, mode of inheritance, clinical presentation for Dyskeratosis Congenita

1) AR (very rare) 2) WRN mutation = RecQ helicase dysfunction (involved in DNA repair) = DNA instability 3) growth retardation (increased collagen), premature aging (like Progeria)

gene, protein, mode of inheritance, clinical presentation for Werner syndrome

deficiency in factor IX protein (coagulation) 1) x-linked recessive 2) AtoG in F9' UTR = increased transcription factor binding = decreased F9 expression

hemophilia B (gene, inheritance, mutation type)

it reaches a GC-rich inverted repeated followed by seven A residues (loop forms a stable loop that leads to termination and dissociation of RNA from DNA template

how does RNA polymerase know when to terminate transcription?

acetylation changes positively charged AA (lysine and arginine) to neutral, thus playing a large role in gene expression

how does acetylation change the composition histone tails?

1) exonuclease activity (proofreading) removes unpaired base 2) correct base reinstered by DNA polymerase

if tautomeric form converts back to normal state, how does DNA polymerase correct this abnormally placed nucleotide?

alternative splicing

joining of exons of a gene in varied combinations (creates multiple RNAs from single gene)

eukaryotic has 3 nuclear (1 in coli) and cannot recognize promoters TFs [mitochondria have their own RNA polymerase that is similar to that of E. coli)

major differences between eukaryotic and prokaryotic RNA polymerases?

1) NFI gene mutation = unstable neurofibromin (downregulates RAS = cancer growth) 2) nonsense mutation 3) AD 4) neurofibromas, cafe-au-lait spots

neurofibromatosis type I (NF1): gene, inheritance, mutation

30nm fiber (very loopy)

nucleosomes associate with each other to form a more compact structure - results from interactions between adjacent histone H1 proteins

1) Chimeric antigen receptor (CAR) attacks overabundant B-lymphocytes 2) pros: 83% remission 3) cons: very expensive, cytokine-release syndrome (inflammation)

pros and cons of gene therapy for Acute lymphoblastic leukemia; how does it work?

telomerase (several units can be synthesized but not all)

reverse transcriptase that synthesizes telomeric repeat sequences at the ends of chromosomes from its own RNA template to avoid loss of genetic material with each duplication

1) HBB gene, B-globin protein =E6V (Glu to Val) 2) missense 3) transversion (GAG to GTG)

sickle cell: gene/protein involved, mutation type, transversion vs. transition

1) 146 BP of DNA wrapped 1.75x around a histone octamer (consists of two subunits each of H2A, H2B, H3, and H4 protein) 2) H1 binds nucleosome to "linker DNA" to stabilize chromatin fiber

structure/organization of a nucleosome (# of BP, subunits)

during protein synthesis

the large and small ribosomal subunits are only active during what process?

1) transitions - like with like (purine with purine) 2) transversion - like with unlike (purine with pyrimidine)

transition vs. transversion point mutations?

protein congregate that contains exonucleases, decapping enzymes, microRNAs that degrade or store mRNAs for future translation

what are P-bodies?

1) helicase - unzips DNA template 2) SSB - prevents strands from reannealing 3) sliding clamp - links strand to DNA polymerase 4) clamp loading protein - loads clamp protein onto strand 5) DNA primase - synthesizes short RNA primers on lagging strand 6) Topoisomerase - introduce short, reversible breaks on one (type 1) or two strands (type 2) [prevent DNA twisting ahead of fork]

what are all the proteins at the replication fork?

code for all machinery needed = LINE-1 (L1); 17% of genome, 1 L1 insertion/150 births

what are autonomous non-LTRs? percent of human genome? incidence of transposition?

1) add dNTPS to 3' hydroxyl group 2) energy needed comes from cleaving incoming nucleotide (phosphate transferred) 3) proofreading prevents tautomeric forms from being inserted (~100 fold efficiency)

what are common characteristics shared by all DNA polymerases (molecule added, energy, safety measures)?

relies on proteins encoded by LINE-1 to move to new genomic location 1) SVA - SINE (Variable number of repeat-Alu element) = .2% 2) Alu - contains Arthrobacter luteus (Alu) restriction endonuclease = 10%, 1 million copies/genome

what are non-autonomous non-LTRs? percent of human genome? incidence of transposition?

1) block binding of estrogen to its receptor; receptor antagonists in breast and agonists in bone 2) tamoxifen - breast cancer treatment and prevention [partial agonist in endometrium = increase endometrial cancer risk] 3) raloxifene - breast cancer prevention (not treatment); reduces/prevents osteoporosis

what are selective estrogen receptor modulators (SERMs)? how do they work? tamoxifen vs. raloxifene?

1) unstable nucleotide forms with different hydrogen bonding properties (C binds with A rather than G) 2) could lead to T to C mutation if not corrected 3) might shift back to normal conformation

what are tautomeric nucleotides?

1) large in peptide bond formation and small in codon-anticodon interaction 2) P = holds tRNA to growing peptide 3) A = holds tRNA to AA to be added 4) mRNA = binds mRNA being translated 5) E = hold tRNA to leave ribosome

what are the large and small ribosomal subunits involved in? what about the four RNA binding sites (P, A, mRNA binding, E)?

1) start = AUG (Met) 2) stop = UAA, UAG, UGA

what are the start and stop codons?

1) segment of DNA that can jump (copy/excise and reinsert) from one location to another 2) DNA - cut and paste 3) RNA - copy-and-paste (DNA to RNA, reverse transcribed into cDNA, and reinserted into genome)

what are transposable elements? DNA vs. RNA?

bacterial RNA polymerase can work without the help of transcription factors (this is NOT the case in eukaryotes)

what does it mean for bacterial RNA polymerase to be DNA rather than TF dependent?

1) eIF-2 = carries GTP and associates with initiator methionyl-tRNA 2) eIF-4 associates with mRNA via 5' cap 3) PABP (polyA binding protein) stabilizes everything

what eIFs and proteins are involved in translation initiation?

1) increase = genetic, drugs, obesity, age of menarche (first period) 2) breast feeding, having kids, exercise

what factors increase risk for breast cancer? prevent it?

a sequence of DNA that specifies an RNA molecule 1) proteins (mRNA), structural (tRNA), enzymatic (ribozymes, rRNA), or regulatory (microRNAs)

what is a gene? what can its RNA code for?

1) two regions in particular are common to most genes 2) each 6 base pair in length and are 10 and 35 bp upstream of transcription start site [having two regions gives enzyme directionality on where to go]

what is unique about the promoter regions in bacteria?

1) snRNA + protein = snRNP congregates to form a spliceosome 2) within the nucleus

what makes up a spliceosomes? where do these operate?

1) introns must be removed 2) UTRs (untranslated regions) have to be processed

what must occur before an mRNA molecule can leave the nucleus?

1) eIF2-GFP hydrolyzes, releasing all other eIFS 2) 60S subunit binds, and elongation begins

what occurs when the eIF and associated complex reaches the first AUG codon?

the looped domains will unravel to 10nm (freer) for higher-level gene expression [in other words, DNA goes into very coiled part when it is NOT replicating]

what significant activity occurs on the looped domains of 30nm fibers?

1) basic amino acids (arginine and lysine) = positive charges neutralize highly negatively charged DNA

what type of amino acids are abundant in histones? what function do they serve on the DNA strand?

1) mRNA = II 2) tRNA = III 3) rRNA = I

which RNAs are responsible for the creation of mRNA, tRNA, and rRNA?

1) safe = strep, erythro 2) dangerous = chloram, tetra, and cyclo (very bad for eukaryotes)

which antibiotics (streptomycin, tetracycline, chloramphenicol, cycloheximide, and erythromycin) are safe to use on humans (that is don't interrupt mitochondria synthesis drastically), and which ones are not?

LTRs (long-terminal-repeat retrotransposons) are HERVs (human endogenous retroviruses) in humans = 8% of genome but are immobile after they insert into the genome

why are LTRs in humans not a threat?

1) flat structure = stacking/packaging of DNA 2) any change distorts the helix (recognized by repair mechanisms)

why are nucleotides' flat structure important? what happens if this ability is compromised?

net force created from hydrogen bonding of base pairs requires a lot of energy to break (even more for molecules with more G-C bonding)

why does any type of DNA replication or RNA synthesis require so much energy?

GTP molecule added in reverse orientation to the 5' terminal for mRNA stability and helps align it on the ribosome during translation

why is a 7-methylguanosine cap added to the 5' end of an mRNA molecule?

polyA polymerase adds ~200 A nucleotides to the 3' end (AAUAAA signal sequence) = increase mRNA stability (longer = more stable) [does not require a template

why is the 3' end of mRNA polyadenylated?

some proteins we need immediately and a lot of (e.g. histones only have 1 exon)

9% of genes have only 1 exon and zero introns (thus cannot participate in exon shuffling or alternative splicing). Why is this important?

group of diseases associated with myelin/axonal protein issues (spectrum of severity depends on size of change) 1) large duplicated PMP22 (point mutation) 2) 1:2500

Charcot-Marie-Tooth: mutation type, gene, inheritance

uses lots of energy (from broken tRNA peptide bond) to precisely (fidelity) attach appropriate amino acid onto the A site of the tRNA

Describe the function of aminoacyl-tRNA synthetase

actinomycin D

Inhibits RNA polymerase in both prokaryotes and eukaryotes (antibiotic and anticancer)

10nm fiber

Nucleosomes connected by stretches of DNA bead formation (~200 base pairs protected from nuclease)

1) Streptomycin: Inhibits p-site 2) Tetracycline: Inhibits a-site 3) Chloramphenicol: Inhibits peptidyl transferase (forms peptide bonds between AAs) 4) Cycloheximide: Inhibits peptidyl transferase 5) Erythromycin: Inhibits eEF2 translocation

Outline how the following antibiotics affect protein synthesis: streptomycin, tetracycline, chloramphenicol, cycloheximide, and erythromycin

HEXA gene mutation = lysosomes open = neurons die 1) sometimes caused by point mutation that alters splicing of mRNA of HEXA gene 2) most common = 4 base insertion causes frameshift 3) Ashkenazi Jewish population

Tay-sachs: gene, inheritance, mutation

polymorphism

The coexistence of two or more distinct forms in the same population (generally considered if over >1% of population has that alternative)

chromatin

The complex of DNA and proteins that makes up eukaryotic chromosomes

rifampicin

antibiotic; inhibits DNA-dependent RNA polymerases (effective against TB, Legionnaires, leprosy (only prokaryotes)