microbio 3

central dogma: _ is transcribed to _, which is then translated to _

DNA; RNA; protein

information stored in DNA (genes) flows from

DNA to RNA to protein

replication is _ copying of DNA

semi-conservative

_ is the creation of RNA from a DNA template

transcription

_ is the creation of protein (polypeptides) from the code on the RNA transcript

translation

semiconservative DNA replication

2 strands separate, each serving as a template for synthesis of a complimentary strand

DNA synthesis is _; each daughter cell obtains one old and one new strand

semi-conservative

biochemical direction of the synthesis (DNA or RNA) goes from

5’ to 3’

in bacteria and archaea, we typically have _ genome

one large circular

DNA in most bacteria is

circular

DNA replication starts at the

origin of replication

180 degrees from the origin of replication

termination point

DNA replication always starts at this one location on the circular chromosome

origin of replication

origin of replication

provides a place where the initial factors recognize the DNA and begin to lay down there and pull it apart

ORI usually _rich

AT (A and T sequences)

why is the ORI AT rich

easier to pull apart and separate

two replication forks go in _ directions to meet at the opposite end of the circle

opposite

role of topisomerase

de-concatenated circular genome into individual circles

eukaryotic systems

big long linear chromosomes

multiple origins of replications

bacteria and archaea only have _ origin or replication(s) that starts at one specific site and goes in both directions, meeting at opposite ends of the circle

one

the origin of replication is _. the species has its own _ origin of replication

specific

if you have plasmids inside the cytoplasm, they’re going to have _ origins of replication

different

one way the cell controls how to replicate plasmids separately/independently from the chromosome

different origins of replication

most plasmids can be replicated _ because they have their own unique origin of replication

autonomously (independent of the chromosome replication)

clone one thing in an organism and transfer it by

change or repeat the origin of replication so that it is compatible with the new species

why e.coli is used as genetic background for all sorts of manipulations

easy, lots of tools available, reliable, lot of strains good for cloning

wont be able to transfer cloned part unless

they include an origin of replication that is compatible with their species

_ replication from a single origin

bidirectional

where DNA is unwound

replication fork

replicon

portion of the genome that contains an origin that is replicated as a unit

ribose has a _ at the 2’ position

OH

deoxyribose has a _ at the 2’ position

H

goes into the RNA

OH at the second carbon position

how to know if its a RNA based molecule or a DNA based molecule

look at second carbon

_ is much more labile (broken apart much easier) because the _ makes it much more _ under heat conditions or under high pH conditions

RNA; OH; reactive/breakable

inside the cell, RNA comes and goes relatively

rapidly

we degrade RNA inside the cell so that new _ can be made = flux of how _ get made in waves because of the fragile nature of RNA

proteins

OH is easy for _ and _ to break apart RNA

enzymes; physiological conditions like heat and basic chemistry

DNA can basically last _ in the environment under dry, stable conditions

forever

2’ _ is _ so RNA is easy to hydrolyze

hydroxyl (OH); nucleophilic

to replicate DNA, you need to have a pool of

nucleotidetriphosphates (deoxyforms of all four DNTPS: ATP, TTP, CTP, GTP)

TP is ATP, GTP, CTP stand for

triphosphate (carrying three phosphates in a row)

triphosphates are _ molecule

highly energized

polymerization process is using the high energy from _ to drive the formation of new bonds to add each base at a time

TP

every time we start a new strand of DNA, we need a _ to get it going

primer

primers are almost always made of _ which means we not only need the four DNA-based nucleotides (deoxyforms), we also need the _ nucleotide TP to fully perform the process of DNA replication

RNA

to make primers: four _-based nucleotides

to make DNA: four _

RNA; deoxynucelotide triphosphates

much more abundant supply of _ during DNA replication, but still need RNA primers

dNTPS

_ catalyzes synthesis of complementary strand of DNA

DNA polymerase

DNA synthesis in _ direction forming phosphodiester bonds

5’ to 3’

DNA polymerase enzymes require

a template (direct synthesis of complementary strand)

a primer (DNA or RNA strand to start polymerization)

dNTPs (dATP/dTTP/dCTP/dGTP: deoxynucleotide triphosphates)

DnaA

initiator protein: initiation of replication=binds origin of replication

helicase

breaks hydrogen bonds holding two strands of double helix together; promotes primase activity; involved in primosome assembly

helicase loader

together with DnaA, directs helicase to DNA template

dna gyrase

relieves supercoiling of DNA ahead of helicase; separates daughter molecules in final stage of replication

SSB proteins

bind single-stranded DNA after strands are separated by helicases

primase (DnaG)

synthesis of RNA primer; component of primosome

DNA polymerase III

catalyzes most of the DNA synthesis during replication

has 3’ to 5’ exonuclease (proofreading) activity

DNA polymerase I

removes RNA primers and fills the resulting gaps in DNA

DNA ligase

synthesizes phosphodiester bonds to join Okazaki fragments

tus

acts as termination of replication

topoisomerase

separates daughter molecules in final stages of replication

unwinds DNA strands = rotational activity separating the double strand into two single strands

helicase

prevent from immediately snapping back together again as double stranded DNA = lock two strands apart temporarily so that the rest of the copying can happen

single stranded binding proteins (SSB)

keeps strands apart for replication to occur

single-stranded DNA binding proteins (SSB)

working upstream of the helicase to prevent the supercoiling = positive supercoiling (reverse twisting action where it clips one of the two strands so that it rotates in the opposite direction and religate/reseal it back together

topoisomerase

topoisomerase works _ of the helicase so that the DNA coils dont get all bunched up on each other and possibly break

in front

DNA primase uses _ as a template

RNA primers

PCR (in vitro version of DNA replication) uses _ primers (oligonucleotide primers that are already pre-synthesized). serve as the template or bind to template and serve as priming piece that gets the replication started

DNA

dna gyrase

changes the structure of DNA by transiently breaking one or two strands without altering the nucleotide sequence of the DNA

primase

synthesizes short complementary strands of RNA primers (10 nucleotides) needed by DNA polymerase

polymerase that does the vast majority of the copying

DNA polymerase III

DNA polymerase III is called the _ because its got multiple functional core enzymes that are all working simultaneously

holoenzyme

three heads (core enzymes) of the DNA polymerase III are used for

copying DNA

clamp loader of DNA polymerase III is used for

positioning it on the DNA

enzyme that makes almost all the DNA: the leading strand, the lagging strand, okazaki fragments on lagging strand

DNA polymerase III

_ does the first round of all the copying of the DNA

DNA polymerase III

fundamentally the most important DNA replication enzyme

DNA polymerase III

e.coli has 5 DNA polymerases with _ playing the major role in replication

polymerase III

DNA polymerase III holoenzyme

complex of 3 core enzymes (each composed of three different proteins) and several other proteins

for DNA polymerase III, _ of the core enzymes replicate one of the DNA strands while _ replicates the other strand

two; the third

we’re making primers primarily on the _ strand

lagging

leading strand is going from _: copying the DNA _ right behind the replication fork

5’ to 3’

since the leading strand is copying DNA from 5’ to 3’

it doesnt need to be interrupted and it can continuously go along that strand because it tucks in right behind the replication fork and thats how it travels (same direction as the movement of replication fork)

lagging strand has functional problem

cant go in the 5’ to 3’ direction directly

since the lagging strand cant go in the 5’ to 3’ direction directly

loops the DNA and makes repeating copies of it

each loop the lagging strand makes is called

okazaki fragment

okazaki fragment

discontinuous short fragments that get made on the lagging strand

each okazaki fragment has its own _ and gets started _

RNA primer; independently

DNA polymerase III makes

the leading strand and the lagging strands at the same time

lagging strand is different from leading strand because

needs lots of primers and restarts of the synthesis

remove the primer and reconnect the broken pieces of DNA

DNA polymerase synthesis is in the _ direction only

5’ to 3’

the lagging strand is synthesized in short fragments called

okazaki fragments

a _ is needed for the synthesis of each okazaki fragment

new primer

holoenzyme moves in the direction of the _ while polymerizing the lagging strand by _ synthesis and the leading strand by _ synthesis

replication fork; discontinuous; continuous

on the lagging strand, we need a new DNA polymerase called

DNA polymerase I

mows out or removes little RNA primers and fills those gaps in with DNA

DNA polymerase I

DNA ligase

seals the gaps. once the polymerase adds DNA right up to the junction of the previous okazaki fragment, there is this little nick/molecular gap that needs to be stitched together

almost entirely on lagging strand (only need to operate once on leading strand and theyre done)

DNA polymerase I, DNA primase, DNA ligase

DnaA proteins bind _ causing bending and separation of strands

oriC (origin of replication)