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DNA replication, RNA transcription, protein translation

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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  


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