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WVSOM - Molecular-1

DNA replication, RNA transcription, protein translation

What are the three types of macromolecular synthesis? DNA replication (DNA synthesis), transcription (RNA synthesis), and translation (protein synthesis)
What is the basic dogma of biology? DNA - RNA - protein
Where does prokaryotic replication take place? In the cytoplasm
Where does eukaryotic replication take place? In the nucleus during S phase of the cell cycle
What is the purpose of DNA replication? Duplicate chromosomes, so that after mitosis each daughter cell will inherit a complete genome
In what type of cells does DNA replication occur? In dividing cells only; non-dividing cells are blocked in G0 and do not progress into S phase, so they do not replicate their DNA
What are the requirements for DNA replication? DNA polymerase, Mg2+, template, primer, and dNTPs
In what direction does DNA replication occur? 5` - 3` direction
What does complementarity mean? For each A on the template strand, there is a corresponding T added to the new strand. The same applies for a G on the template strand. A corresponding C is added to the new strand
What enzyme is reponsible for removing mismatched nucleotides? 3`-5` exonuclease
Bidirectional means? Replication proceeds in both directions from central origins of replication (ori)
The type of replication in which the lagging strand is synthesized Discontinuous replication
DNA in a newly synthesized daughter chromosome contains one new strand and one template strand. This is known as? Semiconservative replication
DNA polymerase does what? It is an enzyme that catalyzes the polymerization of dNTPs into DNA
How many types of DNA polymerases do prokaryotes have? I, II, and III
What are the different types of DNA polymerases in eukaryotes? Pol-delta, pol-alpha, pol-beta, pol-gamma
Pol-delta: location, function, processivity, proofreading, use of RNA primer Location: nucleus; function: leading strand synthesis; processivity: >100,000 bp; proofreading: yes; use RNA primer: yes
Pol-alpha: location, function, processivity, proofreading, use of RNA primer Location: nucleus; function: lagging strand synthesis; processivity: ~180 bp; proofreading: no; use RNA primer: yes
Pol-beta: location, function, processivity, proofreading, use of RNA primer Location: nucleus; function: fill in gaps for repair; processivity: ~20 bp; proofreading: no; use RNA primer: no
Pol-gamma: location, function, processivity, proofreading, use of RNA primer Location: mitochondria; function: synthesis of both strands; processivity: ~8,300 bp; proofreading: yes; use RNA primer: no
The cofactor that is required for DNA polymerase activity? Mg2+
The pre-existing strand read by DNA polymerase is known as? The template strand
What are the building blocks of DNA? dNTPs (deoxynucleotides) = dCTP, TTP, dGTP, dATP
What is significant about dNTPs? They lack a 2` hydroxyl group
How is thymine different from uridine? Where is uridine used? It is methylated at the 5` position; uridine is used for RNA
Where does nucleotide polymerization get its energy from? How is this energy stored? Hydrolysis of the triphosphate bonds; energy is stored in the triphosphate bonds as electrostatic repulsion of their negatively charged oxygens
T/F: DNA polymerase can bind a 5` phosphate with an incoming 3` -OH? F; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide
What is a primer? It is a strand with a free 3` -OH group; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide
What direction does DNA synthesis proceed in? 5` - 3` direction!!
What direction is the template strand oriented? 3` - 5`; it is antiparallel to the newly synthesizing strand
Which DNA polymerases have the ability to proofread and determine if the new nucleotide is complementary to the corresponding base of the template? Pol-delta and pol-gamma
What happens if an incorrect nucleotide is incorporated? The polymerase's 3`-5` exonuclease activity "kicks back", excising the mismatch; pol-alpha and pol-beta lack this activity
The leading strand Continuously synthesized in the 5` - 3` direction
The lagging strand The opposite strand synthesized; antiparallel to leading strand; discontinuously synthesized in 5`-3` direction; stretches known as Okazaki fragments
How long are Okazaki fragments? 100-200 bp
What does semiconservative mean? It means that each chromatid receives one de nova and one parental strand; DNA replication is always semiconservative, never conservative nor non-conservative
What is the origin of replication (ori)? It is the sequence where DNA replication begins
How many ori's do prokaryotic chromosomes have? How many do eukaryotic chromosomes have? Prokaryotes = 1 ori per chromosome; eukaryotes = multiple ori per chromosome
What is the general procedure of DNA replication? Factors recognize and bind specific ori sequence, unwind DNA, attract components of replication apparatus, apparatus moves down chromosome (away from ori), replicate DNA as it goes; DNA replication is bidirectional, two apparatuses assemble around ori
What is a replicon? What is their average length? Region of eukaryotic chromosome that is replicated as a unit, from one central ori; length is about 200 kb (this is about the length of DNA loops anchored to scaffold proteins, suggesting that these proteins may be involved in defining replicon length)
Why is it a benefit to have multiple replicons? So that different regions of the genome can be replicated simultaneously; replication begins at the ori in the center of the replicon and extends in both directions until it reaches the end of an adjacent replicon
What is the purpose of helicase? Enzyme that unwinds DNA; one of the first factors to bind ori; serves to open double helix so DNA polymerase can replicate strands; is part of replication apparatus
What are single stranded bindng proteins (SSB)? Factors that stabilize single stranded DNA by preventing it from winding back into double helix
What does the DNA replication apparatus consist of? Helicase, DNA polymerase-delta, pol-alpha, beta-clamp, and primase; two apparatuses assemble, one on each side of the ori, and each moves in the oppostie directions, replicating DNA as they go
What does the beta-clamp do? Ring-like protein that wraps around DNA to stabilize association of replication apparatus; required for pol-delta processivity; without clamp = pol-delta only replicates short oligonucleotides (< 200 bp), with clamp = stretches > 100 kb are produced
Strand that is continuously synthesized, in the 5`-3` direction? Leading strand
What is DNA polymerase-delta? Enzyme that replicates leading strand, reads template one base at a time, incorporates complementary nucleotides and ligates their 5` phosphate to the 3` -OH of the growing strand
What is the overall synthesis of the lagging strand? How is its synthesis overcome? Overall synthesis is in the 3`-5` direction (wrong direction); its synthesis is overcome by synthesizing discontinuous short stretches called Okazaki fragments
What does primase do? Enzyme that binds unwound lagging strand and transcribes short stretches of RNA (< 15 bp); RNA serves as primer, providing 3` -OH group required by pol-alpha
What is the purpose of DNA polymerase-alpha? Enzyme uses RNA primer to synthesize Okazaki fragments of the lagging strand; NO proofreading ability; only synthesizes one fragment at a time while helicase contiues to unwind DNA for next fragment, so lagging strand held in loop
What happens when pol-alpha reaches a primer at the end of a previous fragment? Lagging strand is released, primase makes next primer at end of new single stranded region, process is repeated
What are the three enzymes involved with removal of RNA primers from the lagging strand? RNAase, DNA polymerase-beta, DNA ligase
What does RNAase do? This enzyme digests any RNA; in replication it serves to remove lagging strand primers
What does DNA polymerase-beta do? It fills in the gaps in DNA (~ 20 bp); in replication it fills in the gaps left after RNA primers are removed; N.B. = DNA polymerases can only add nucleotides, they cannot link DNA fragments (pol-beta leaves nicks in DNA)
What does DNA ligase do? Enzyme that binds any free 3` -OH and 5` phosphates of DNA; in replication it seals the nicks between Okazaki fragments left by pol-beta
What happens as a result of helicase unwinding? What enzyme helps fix the problem? Supercoiling increases; topoisomerases helps to restore DNA to its proper level of supercoiling
What happens when the last RNA primer is removed from the end of the chromosome? It leaves an overhang that cannot be filled by DNA polymerase; if this didn't get resolved, then every time dividing cells replicated their DNA, the lagging strands would lose a bit of their telomeric sequence, leading to chromosomal destabilization
What enzyme is responsible for filling in the gap left when the last RNA primer is removed? Telomerase (it is a type of reverse transcriptase) - it fills in the gaps as well as extends the length of the telomere
What are some interesting facts about telomerase? Activity and length decreases with age; primary cell culture lines have no telomerase activity; immortalized cell culture lines divide indefinitely b/c of activated telomerase; activated in cancer cells and believed to contribute to immortalization
Where is gene expression controlled? Pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels
What does transcription mean? RNA synthesis; RNA's often called transcripts and are said to be transcribed
What are the three classes of RNA? Messenger RNA, ribosomal RNA, and transfer RNA (small nuclear RNA)
Messenger RNA: abbreviation, function, abundance, RNA polymerase mRNA, encodes polypeptide sequences, lowest, pol-II
Ribosomal RNA: abbreviation, function, abundance, RNA polymerase rRNA, components of ribosomes, highest, pol-I
Transfer RNA: abbreviation, function, abundance, RNA polymerase tRNA, AA carriers, moderate, pol-III
Small nuclear RNA: abbreviation, function, adundance, RNA polymerase snRNA: splicing, moderate, pol-III 5S rRNA: ribosomes, moderate, pol-III
What percentage of rRNA makes up the total RNA? 90%
T/F: There is the same amount of tRNA as rRNA T; there appears to be less b/c their mass is much less than rRNA
Upstream and downstream Specify region of gene relative to base where transcription starts; upstream = 5` and downstream = 3`; during transcription, new RNA strand synthesized in 5`-3` direction; downstream = direction of transcription; upstream = front of gene
What number is assigned to transcription start site? +1; upstream bases are negative, downstream bases are positive
What is the promoter? Region of DNA used to activate / repress transcription of gene; position of promoter cannot be moved relative to gene; pol-I and pol-II promoters usually adjacent to gene on upstream side; pol-III promoters within gene
What is an enhancer? Region of DNA that regulates transcription (like a promoter)
What is unique about an enhancer? Can move relative to gene it controls; 5`-3` orientation can be flipped; distance between enhancer and gene can be altered; enhancers often located great distances from genes (> 40 kb)
What is a minimal promoter? Smallest region of full promoter that will drive detectable transcription (i.e. priming transcription); drive constitutive, low level transcription; not responsible for regulation
TATA box? Most minimal promoters for pol-II; possesses sequence TATA(A/T)A
TATAless promoters? Promoters that lack any sequence similar to TATA box; genes tend to be expressed at low levels
AT rich sequences Adenosine and thymidine = 2 bonds; held more weakly (compared to GC areas); property may facilitate strand separation during initiation of transcription
The initiation complex Cluster of proteins known as transcription factors, that assemble around promoter to initiate transcription
General transcription factors (GTF) Proteins that form core of initiation complex (at miniml promoter); called general to distinguish them from upstream transcription factors (UTF) responsible for gene specific regulation; UTF bind GTF to increase size of initiation complex
TATA binding protein (TBP) General transription factor that bind TATA box; other general transcription factors assemble around TBP to form core initiation complex at minimal promoter
Start of transcription Usually a purine (A > G); 26-34 bp downstream from TATA box; mammals = consensus around (G/A) start is GTTGCTCCT(G/A)AC
What is the mechanism of action for the initiation of mRNA transcription? TBP first binds several general transcription factors in nucleoplasm; complexes then find TATA box; other general transcription factors recruited to form core initiation complex; complex attracts RNA pol which "looks" for purine downstream (~ 30 bp)
What is the purpose of upstream promoters and enhancers? Required to elevate and regulate transcription
What are recognition sequences? Promoters and enhancers bound by transcription factors; short (4-10 bp); palindromic (inverted, end to end repeats); can vary from gene to gene (consensuses)
Is the TATA box a recognition sequence? Yes; for TBP; most common
Name two other (besides TATA box) recognition sequences CCAAT box - 2nd most common; raises baseline transcription; bound by TF CP1 (CCAAT binding protein 1) SP1 recognition sequence - 3rd most common; consists of GC rich regions (20-50 bp); bound by SP1 TF; common in TATAless promoters
NF-1 TF that binds to similar CCAAT sequences; mutated in patients with neurofibromatosis 1; contribute to frequency of CCAAT boxes in genome
Transcription factors (TF) Proteins that bind recognition sequences to control transcription; can be activators or repressors
DNA binding domain Region of TF that recognizes and binds specific sequence of DNA; usually basic (+ charged); charge forms ionic bonds with negatively charged phosphates on surface of DNA
Activation domain Region of TF that induces RNA synthesis by attracting RNA polymerase; can be on either side of DNA binding domain; position varies even within same family of TF
Acid Blob Model: activation domains as masses of negative charge with little structure and few specific interactions - list the four observations 1. Variable (random sequence of negatively charged AA) 2. No specific structures (random coils) 3. Attract same RNA polymerase 4. RNA pol trageted to location due to attraction to negatively charged DNA
What happens as TF bind the recognition sequences of promoter and enhancer regions? Sequences become grouped together on DNA; "stick" to each other via ionic and hydrogen bonds; upstream TF contribute to initiation complex by bind general TF at minimal promoter; initiation complex becomes large mass of negative charge; attracts RNA pol
What are the four TF families? Classified based on DNA binding domains; Helix-turn-helix (HTH) family, zinc fingers, helix-loop-helix (HLH), and leucine zippers
Helix-turn-helix (HTH) family DNA binding domain = 2 alpha helices positioned at right angles to each other, connected by short linker region; 1 alpha helix has basic face (binds DNA by wedging into major groove)
What is another name for the helix-turn-helix (HTH) DNA binding domain? Homeobox, after the first class of TF where it was identified (the homeotic genes of the fruit fly Drosophila)
Zinc fingers DNA binding domain = cysteine and histidine residues chelated to central zinc ion (creates loops in polypeptide), loops act like fingers wedging themselves into major groove to bind DNA
What are the three subfamilies of zinc fingers? C2H2 zinc finger class, C4 class, C6 class
C2H2 zinc finger class A pair of cysteines (2-4 AA apart), a linker region (~ 11 AA long), a pair of histidines (3-4 AA apart)
C4 zinc finger class Like the C2H2, except that there are two pairs of cysteines with no histidine
C6 zinc finger class Three pairs of cysteines
Give two examples of helix-turn-helix (HTH) TF Hox genes and PIT-1
Helix-loop-helix (HLH) DNA binding domain - similar to HTH; 2 alpha helices separated by linker region; linker region are longer; no sequence similarity between HLH and HTH factors
Are HLH factors heterodimers? What does that mean? Yes; it means that two different polypeptides must bind to each other before they will bind DNA
What are the HLH factors? Positive, negative, and ubiquitous; negative and positive factors compete with each other for association with ubiquitous factor; heterodimers with negative factor = repress traget genes; positive/ubiquitous heterodimers = activate transcription
What is an example of a helix-loop-helix (HLH) factor? MyoD
rRNA Majority of total RNA
Cell strategies for generating rRNA abundance Includes multiple copies of rRNA genes (intermediate sequence class); multiple transcripts to be simultaneously produced from each gene (known as Oscar Miller feathers)
Nucleolus Region where ribosomes are produced; not membrane bound; region where multiple rRNA transcripts expressed from multiple genes w/ protein binding each transcript
How many rRNA's does each gene encode for? 3
What transcribes the 3 rRNA's? Transcribed by Pol-I
What are the transcribed rRNA's known as? pre-rRNA
28S, 18S, 5.8S rRNA's after the intervening sequences are cleaved away (from the pre-rRNA)
5S Fourth rRNA transcribed from separate gene, 5S rRNA; transcribed by Pol-III (same pol that produces tRNA)
Ribonucleoproteins Proteins that assemble around rRNA to form large and small subunits of ribosomes; ribosomes = RNA + protein; proteins begin association in nucleus while rRNA is being transcribed (represent major component of nucleolus)
tRNA folding Internal annealing that fold tRNA's into clover leaves; D & T(psi)CG loops also fold back on each other to generate L-shaped structures
Anticodon Nucleotide triplet at apex of tRNA; complementary to specific codon of mRNA; in ribosomes, anticodon binds codon; this is how codons specify AA
Do tRNA and rRNA promoters have TATA boxes? NO; however, TBP still component of initiation complexes
RNA pol-III promoters Within their genes! DNA folds back on itself to form internal bp; small RNA's transcribed by pol-III characterized by internal annealing; pol-III recognizes same tertiary structure in DNA and RNA
mRNA, tRNA, ribosomes All produced in nucleus
Where does translation occur? Cytoplasm
How do factors get in / out of nucleus? Nuclear pore complexes; 16X larger than ribosomes; made of 30 different proteins (known as nucleoporins); transport of factors mediated by importins and exportins (bind specific factors, shuttle them through nuclear pore)
tRNA charging tRNA binding with specific AA; aminoacyl-tRNA synthetase binds 3` -OH of tRNA with carboxyl group of AA; rxn requires ATP
How many tRNA's are there? > 50 (but only 20 AA); this means that several tRNA's bind a given AA; however, each tRNA can bind one AA and one codon
What contributes to the degeneracy of the genetic code? Multiple tRNA's for a given AA
3 bases in mRNA Code for an AA
Codon A nucleotide triplet that specifies a particular AA
Characteristics of the Code Triplet, unambiguous, degenerate, wobble, colinear, non-overlapping, universal
Triplet Every 3 bases of the mRNA sequences, read 5`-3`
Unambiguous Codons only specify 1 (ONE) AA
Degenerate Most AA specified by several different codons; 4 nucleotides (A, U, C, G) can form 64 triplet combinations; however, only 20 AA; therefore, AA must be encoded by multiple codons
Wobble For different codons that specify same AA, it is usually 3rd base that varies; first 2 are usually identical
Colinear Codon sequence is parallel to AA sequence
Non-overlapping Next codon begins where last one ends
Universal All living organisms on earth use same code
Start codon AUG; codes for methionine; all polypeptides begin with Met; note = amino terminus can be cleaved to remove Met; ribosome 1st grabs mRNA, reads down length in 5`-3` direction looking for start codon; when it finds AUG, it starts to add AA to growing chain
Stop codons UAA, UAG, UGA
Translation Protein synthesis
Polypeptide Polymer (chain) of AA; proteins are polypeptides or composites of different polypeptides
Peptide bond Carboxyl moiety of 1 AA is bound to AA moiety of next
Residue AA in sequence of polypeptide
Directions During translation, RNA read in 5`-3` while polypeptide synthesized form amino to carboxyl ends
Stages of translation Initiation, elongation, release
Accessory factors promoting stages of translation Initiation factors (IF), elongation factors (EF), release factors (RF)
Initiation stage Ribosomes assembled with mRNA between large and small subunits along with 1st aminoacyl tRNA (Met); tRNa bound to P site of ribosome
Elongation stage Addition of AA to growing polypeptide chain
Peptidyl site (P site) Region of ribosome that binds tRNA that is attached to growing polypeptide
Aminoacyl site (A site) Region of ribosome that bind aminoacyl tRNA for next AA to bind growing polypeptide
How does a polypeptide form? Codon on mRNA reaches A site; ribosome looks for aminoacyl tRNA with complementary anticodon; anchors bp together; polypeptide at P site transferred to AA at A site (forms peptide bond); mRNA moves from A site into P site
Release stage Completed polypeptide, mRNA and both ribosomal subunits all come apart
Protein sorting Process of targeting proteins to specific compartments (i.e. mitochondria, chloroplasts, peroxisomes, RER)
Signal sequence Targeting protein to specific compartment; newly translated region on protein that is bound by docking protein, which in turn binds receptor on organelle's membrane (allows protein to translocate thru channel)
Protein sorthing and signal sequence in RER Docking protein binds signal sequence while polypeptide stil being translated; docking protein then binds transmembrane receptor, anchoring ribosome to RER; channel = translocon; translation continues, polypeptide translocated in lumen of RER
What is unique about transmembrane receptors? They have hydrophobic regions called stop transfer sequences that embed in hydrophobic core of bilayer when released from translocon after translation and translocation of protein is complete
Intercellular membrane trafficking Transport of protein to its final destination (i.e. plasma membrane, lysosome, extracellular secretion) after being deposited in specific compartment (i.e. RER)
Protein movement through RER Vesicles move from ER to cisternae of golgi towards plasma membrane by budding of one compartment and fusing with the next; proteins modified as they go
What happens to vesicles when leaving the golgi? Some become lysosomes; other fuse with plasma membrane (transmembrane proteins become integral proteins of plasma membrane - i.e. receptors or transport proteins); proteins in lumen released into extracellular space for secretion
Protein orientation As passage of protein through intracellular membrane continues, if amino end of protein faces lumen of ER, it will continue to face lumen's of vesicles and golgi; if vesicle fuses with plasma membrane, the amino end with face extracellular space
Proteins free in lumen of transporting vesicle If vesicle fused with plasma membrane will be deposited into extracellular space
Proteins facing cytoplasm of transporting vesicle If vesicle fused with plasma membrane, then protein will continue to face cytoplasm throughout intracellular membrane system
Lumenal side of ER, golgi, vesicles, exoplasmic side of plasma membrane E face
Cytoplasmic side P face
Five principle types of post-translational modification Disulfide bond formation, glycosylation (+ carb side chain), cleavage, polypeptide folding, multisubunit assembly; all occur in ER (glycosylation also occurs in golgi; cleavage also occurs in golgi & vesicles)
Proper protein folding is mediated by Helper proteins, chaperons
Leucine zipper transcription factor examples Fos, Jun, Myc, C/EBP, CREB
Leucine zippers Dimeric proteins (homodimers - Myc; heterodimers - Fos, Jun)
Interesting characteristic about leucine zippers Interaction domain; region of protein that specifically binds another protein
Interaction domain of leucine zipper Leucine zipper protein = a-helix w/ leucine every 7 residues; 3.6 AA/turn; leucine every 2 turns; leucines aligned on same side of helix; leucine = hydrophobic; alignment = hydrophobic face; faces of 2 zippers binds = dimer
DNA binding domain of leucine zipper Extensions of a-helices which grip recognition sequence on each side of DNA ("scissors grip" binding)
mRNA processing: primary transcript RNA's that have not been processed; processing commences before transcription is completed, so two occur simultaneously
mRNA processing: capping Guanosine added to first nucleotide of primary transcript by unusual 5`-5` bond; cap methylated (also 2` -OH of 1st & 3rd nucleotide); 3 sites methylated with all mRNA's; capping & methylation = increases mRNA stability (prevents degradation)
mRNA processing: splicing Removal of section of primary transcript; called intron (> 10,000 bp long); retained regions = exons
Spliceosomes Splice out introns; small complexes of snRNP's, composed of proteins + snRNA's; enzymatic activity in RNA, NOT protein
snRNP's Small nuclear ribonucleoprotein particles
snRNA's Small nuclear RNA's; transcribed by pol-III (same pol that produces tRNA)
Polyadenylation Final event of mRNA that adds 20-300 adenosines to 3` end of transcript; known as poly A tails; believed to increase stability
Polyadenylation signal sequence AAUAAA; transcription does not end at this site; continues for another 500-2000 bp before pol-III fall off DNA; excess RNA cleaved at CA (10-30 bp downstream of AAUAAA)
Poly A polymerase Produces poly A tail by adding adenosines to 3` end of transcript; vast majority of mRNA's are polyadenylated
Example of mRNA that is not polyadenylated Histones
Created by: JaneO
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