DNA

Question	Answer
What does DNA synthesis requires?	1) requires all 4 dNTPs (dATP, dTTP, dCTP, dGTP) 2) DNA polymerase 3)some DNA (DNA template in vitro) 4)RNA primer (or engineered primer in vitro) 5)Mg+
Function of a DNA polymerase I?	1) Enzyme needed for DNA synthesis 2)Catalyzes polymerization of nucleoside into DNA chain 3)An example of a DNA polymerase is the Taq 4) DNA polymerase catalyzes formation of phosphodiester bonds 5)DNA polymerases adds the correct complementary base 6) DNA polymerases catalyze formation of DNA chain from dNTPs
DNA replication (chain, nucleotides, bonds)	1) CHAIN act as a primer 2)new NUCLEOTIDES added to the 3' OH group of the sugar 3) 3' OH group BONDS to phosphate group of new nucleotide (release 2/3 of P in bond)
Function of the 5 known polymerases?	a) 1 and 3 needed for replication from 5' --> 3' b) 1, 2, 4 and 5 needed for repair c) 1 and 3 can remove nucleotides= proofreading
What are the general requirements for all DNA polymerases?	Add new bases to 3' end of existing strand, to synthesize from 5' to 3' direction, and require an RNA primer
Name the 2 types of polymerases and their function	RNA polymerase makes primer and DNA polymerase extends the primer
How does proofreading work?	Polymerase 1 and 3 remove nucleotides going from 3' to 5' direction by making the polymerase to back up, remove error and then moves forward again.
Mutagen	Any agent that increases the number of mutations above background level such chemicals and radiation
What are the three steps of polymerization?	1) Initiation 2) Elongation 3) Termination
Characteristic of PROKARYOTIC replication?	(E. Coli model) It has a single circular molecule of DNA, the replication begins at one origin of replication and proceeds in both directions around the chromosomes
INITIATION step during prokaryotic replication?	1) Origin (= it is a specific sequence and in bacteria it has only one 1 origin) is the location where the DS DNA becomes SS (Replication bubble denatured) 2) Replication is bidirectional 3) It will have two replication forks (= the location of untwisting DNA)
What is the replicator sequence of E. coli?	oriC; the initiator protein DnaA binds to replicator
Difference between a primer and a template	1) A primer is a short nt sequence bound to template strand 2) A template is a strand on which new strand is created and base paring is used to match up correct nt
What is the problem one encounters during the elongation problem?	The problem is that DNA can onlybe synthesized rom 5' to 3' as the replication for advances. In the leading strand (direction is -->; here it goes from 5' to 3') there is no problem but there is one with the lagging strand ( here it naturally goes from 3' to 5' thus the direction has to be from 3' to 5')
What are the basis for a semidiscontinous replication?	1) Partial opening of helix forms replication fork 2) DNA primase is a an RNA polymerase that makes RNA primer; RNA will be removed and replaced with DNA 3) DNA gyrase (a topoisomerase) relaxes supercoiling tension 4) DNA can only synthesize from 5' to 3' direction
General characteristics of Leading strand	1) A single priming event 2) Strand extended by DNA pol 3
General characteristics of lagging strand synthesis	1) Discontinuous synthesis by DNA pol 3 2) there are multiple priming events 3) RNA primer made by primase for each Okazaki fragment
Okazaki fragments	Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication. They are complementary to the lagging template strand, together forming short double-stranded DNA sections
Explain, in general, how does the elongation of the lagging strand works?	1) DNA polymerase I and DNA ligase join Okazaki fragments 2) DNA polymerase I digest RNA primer, adds nt to 3' end 3) SS nick is left between fragments joined by DNA ligase
Explain replication in Eukaryotes base on E. coli	1) Like E. coli DNA unwinds to SSat replication fork and there is a bi-directional replication 2) Unlike E. coli it has many origins of replication, chrs are linear, and there are multiple chrs
Why is it elongation in Eukaryotes more complicated?	They are less known than prokaryotes and have 15 or more DNA polymerases, their mitochondria and chloroplast have own polymerases and there are other polymerases involved in repair.
Describe what is a replicon/replication unit	It is a stretch from origin to terminus, between two replication forks
Why does the E. coli takes less time to replicate its genome than the humans	This is because E. coli has one replicon wheres the human has 10K or more replicons
What happens at the ORC (Origin Recognition Complex) during the initiation of replication in eukaryotes?	The initiating porting binds to DNA and activates when cell enters S growth phase (synthesis).
Identify and explain the most common polymerases used during elongation in Eukaryotes	1) Pol alpha (primase): makes RNA primer for a new strand, adds initial DNA nt 2) Pol epsilon: synthesizes leading strand 3) Pol sigma: synthesizes lagging strand
Briefly explain what are the Telomeres	1) Telomeres are special structure found on the ends of Euk. chrs. 2) They protect chr ends from nucleases and maintain the integrity of linear chrs 3) There is a radial shortening of chrs with each round of cell division and the last section of the lagging strand is unable to replicate. However, the telomeres provide a buffer against gene loss. 3) Telomerase adds extra repeats to 3' end of chrs; this extra repeat is non coding sequence to provide template 4) Telomeric sequence are species- or taxon-specific
What is a TelomerASE	1) enzyme that makes telomere sing internal RNA template 2) made ob both RNA and protein 3) contains RNA template sequence for the telomere
Why is it important to have telomerase?	Despite the fact that telomerase is developmentally regulated and it is related to aging in complex ways without telomerase, the genome continually shortens leading to death.
Definition of a genomec	Total genetic material of an organism a) Viruses: RNA or DNA, one or more pieces, single or double strand b) Prok: Usually one signal chrs., DNA c) Plasmid: Additional smaller autonomous chrs. in Prokaryotes d) Euk: mitochondria and chloroplast have own genomes, main genome is usually in multiple chrs nucleus.
Compare Euk. and Prok. Genomes	Prok. have Nucleoid, are haploid, circular, 1 piece, predominantly DNA, 1 million base pairs (E. coli) Euk have a nucleus , are diploid (most higher Euk), are linear, have multiple chromosomes, 50% consist of protein and have 6 million base pairs (human)
Technique used by DNA to fit the long stands	Supercoiling, it compacts the DNA by over-winding
DNA packing in prokaryote	-Supercoiling caes DNA to become compact -Negative supercoiling is the untwist of the helix (under wound) -Positive supercoiling is the addition of twist in the helix (overwound)
DNA packing in Eukaryote	1) Topoisomerases are enzymes that control coiling. Type one 1 cuts one strand and type 2 cutes both strands 2) Chromosomes are 50% protein d helo to fold up the DNA fit the cell 3) Without packing, a human cells' DNA would be over 2m long
Chromatin	DNA + protein complex in cell nucleus
Histones	Part of chromatin, bind to DNA, and also on-histones with roles in replication, repair, etc.
Nucleosome	Chromatins 'beads' are considered to be nucleosomes. Nucleosomes are a core of 8 histones wrapped with DNA (DNA wraps around 1.65 times which makes the compact DNA)
What are the characteristics of having a chromosome scaffold	It makes the chromosome shorter and fatter than single DNA strand
Difference between transcription and translation	Transcription is the single stranded RNA copied from DNA Translation is the mRNA translated into amino acid sequence (the synthesis of protein)
Difference between DNA and RNA	DNA is double stranded, has thymine, TTP and H (@ 2' C) RNA is single stranded, has uracil, UTP and OH group (@ 2' C)
Difference between RNA and DNA synthesis	RNA Synthesis: only part of the genome synthesized at time (gene expression) and RNA polymerase can begin a new stead DNA synthesis: Double strand requires features like Okazaki fragments and DNA polymerase requires primer for new strand
Identify and define the 4 main types of RNA	1) mRNA: transcript of protein-coding gene (the translation of mRNA produces polypeptides) 2) rRNA: ribosome= rRNA +ribosomal protein 3) tRNA: escorts a.a.'s to ribosomes for translation 4) snRNA: used with proteins in Eukaryote RNA processig
Function of Gene regulatory elements	Regulate transcription of genes
Function of RNA polymerases	Catalyzes transcription from DNA template
In what direction is RNA synthesized and how are DNA template read	RNA is synthesized from 5' --> 3' DNA template is read 3' --> 5'
What is the contemplate strand?	The complementary DNA strand in the DS DNA
Explain the transcription in prokaryotes	It has 3 parts of gene: 1) Promotor: The upstream of the gene. The code tells RNA polymerase where to start & which strand. Promotor and gene are strand specific (E. coli has CONSENSUS SEQUENCES for most promoters meaning that it has the same sequence at -35 (bp) region and -10 (bp) region) 2) The coding sequence of a gene: In bacteria there are no introns (the non coding section of a strand vs. Exons) and is transcribed RNA polymerase (=transcript) 3) Terminator: Code for STOP transcribing
Initiation process in Prokaryotes	1) Only 1 RNA polymerase in bacteria. It has a holoenzyme (=5 subunits of RNA Pol, including sigma factor (= a key to promoter specific-biding needed only for initiation) 2) A closed promoter complex: A holoenzyme bound to helix of ds DNA 3) An ope promoter complex: after holoenzyme as untwisted DNA helix 4) Different forms of sigma factor bind to different promoter sequences OR bind less/more strongly & produce less/more transcript and causes a differential in gene expression
Elongation process in Prokaryotes	1) After transcription begins, sigma factor is released and new nt added 5' --> 3' 2) There is a proofreading process: 2.1) back up 1 NT as in DNA synthesis (backup space style) 2.2) back up 1 or more NT, cleave off errors, resume
Termination process in Prokaryotes	1) Type 1 ends with a string of A's (on DNA) --> U's (on RNA) 2) Type 2 (Rho-dependent): Rho protein bind to C-rich DNA region. Rho unwinds new RNA from DNA template 3) There is no nucleus, thus translation can begin immediately
difference between introns and exon	Introns are the intragenic regions whereas the exon are the expressed regions in DNA and RNA
Differences in the Eukaryotic Genome (introns and Exon)	Introns and exon are in the ds DNA and ss RNA (pre-mRNA). The mRNA contains only the exon which has been translated into protein. The alternative splicing possibilities of mRNA let us see that not every Exon will be in the mRNA but variation exists from time to time
Characteristics in Eukaryotes	1) RNA made in nucleus and protein in cytoplasm. Transcription and Translation happens in different locations. There is an RNA processing 2) There are three RNA polymerases (instead of 1). RNA pol 1(in nucleolus, catalyzes synthesis of 3 rRNA), RNA pol 2(in nucleoplasm, synthesizes mRNA, snRNA), and RNA pol 3 (in nucleoplasm, synthesizes tRNA, 1 rRNA, snRNA). There are additional RNA pols outside nucleus. 3) Different promoter regions, including less direct enhancer begin upstream ~ 200 bp. 3.1) It needs a core promoter (= a sequence needed for RNA synthesis to begin at the right place, begins less than 50bp from start, the initiation sequence (Inr) over the start site) and a cis-acting (= acts ONLY in same molecule vs. trans-acting). There is a TATA box (-30bp, always 5' TATAAAA 3') 3.2) Promoter-proximal element: out 50-200 bp upstream from start which strongly increase initiation but not required 3.3) Activators: regulatory proteins type of promotor-proximal. They are involved with the efficiency of initiation and may function in only certain cell types. 3.4) Enhancers: the type of promoter-proximal element. Either up/downstream , 1000 bp away. Required for max transcription. 4) 5' cap is an Uuusual structure enzymatically added to 5' end made of methylated guanine, protects mRNA and helps bind ribosome. 5) 3' polymerase A tail: 20-200 A's added enzymatically to 3' end. It is added AFTER transcription and protects mRNA from degradation. 6) RNA transcript is not mRNA (called hnRNA or pre-mRNA). Precursor mRNA (pre-mRNA)= pre-processing which includes introns.
Transcription factors?	1) Proteins with DNA binding domains 2) regulate transcription & gene expression 3) bind to promotors and enhancers
Spliceosome?	1) It is the pre-mRNA + snRNPs 2) it contains sn ribonucleic proteins particles (snurps), snRNP (=protein + snRNA) 3) It binds RNA, loop it back, cut and paste (some RNA can self-splice, RNA is the enzyme= Ribozyme)
RNA editing	post-transcriptional change of nt, insertion or deletion
General differences between Eukaryotes and Prokaryotes	1) RNA made in nucleus, protein made in cytoplasm 2) 5 different mRNA polymerases (instead of 1) 3) Different promoter regions, including less direct enhancers 4) 5' cap: The unusual tructure enzymatically added too 5' end 5) 3' Poly A's tail: 20-200 A's added enzymatically to 3' end 5) RNA transcript is not mRNA (called hnRNA or pre-mRNA)

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	Created by enrique.salanic about 10 years ago