Exam 1 - ch 1, 7 Flashcards
memorization
operon
cluster of genes under the control of one promoter, ex. LAC (lactose)
when bacteria is exposed to lactose, the LAC operon activates, regulated by a feedback loop
post-translational modification
when the genetic sequence is edited AFTER translation
model organism
discoveries about model organisms are often true for all organisms
features: small size, small genome, short repro. time, large # offspring
mendel
1856-63, discovered genes through pea plant experiments
Thomas H. Morgan
1910, discovered genes were located on chromosomes
Tatum + Beadle
1941, developed “1 gene 1 peptide” hypothesis
James Watson + Francis Crick
1953, discovered double helix of DNA, won Nobel
Used Rosalind Franklin and Maurice Wilkins’ data
Used ball and stick models, paired A-T and C-G
Crick discovered…
central dogma 1958
Jacob + Monad
1961, determined that enzyme levels are controlled by feedback mechanisms
Nirenberg, Khorana, Brenner, Crick
1961-1967, crack genetic code, working with codons - worked out which codons were which
Sanger, Gilbert, Maxam
1977, invented how to determine nucleotide sequences of DNA
requirements for genetic material
- store info
- transmitted genetically
- replicated when transmitted down
- variable to account for phenotypic variation
Frederick Griffith
1928
experiments with mice
living type S injection
mice died, live bacteria recovered
living type R injection
mice survived
dead type S injection
mice survived
dead type S and living type R
mice died, living type S recovered
cause of mice dying?
DNA from dead S cells escaped lysed capsules and entered R cells by horizontal gene transfer
Avery, MacLeod, McCarty
- 1940s, cemented DNA as genetic material
- experiment involved eliminating DNA, proteins, or RNA
- concluded that transformation only occurred when DNA is present → DNA is the genetic material
process of Hershey + Chase experiment
- used 32P to label DNA, 35S to label protein
- saw that bacteriophage DNA entered the target cell, not protein -> DNA is genetic material
Hershey + Chase
- provided evidence that DNA is the genetic material of T2 phage
- used radioisotopes to distinguish DNA from proteins
Friedrich Miescher
1869, first identified DNA from pus in surgical bandages, called it nuclein
DNA structure
- large macromolecules
- nucleotides form repeating unit linked to form linear strand, two strands form double helix
- 3D structures caused by bending and folding
chromosomes
DNA wrapped around histone proteins
most common form of DNA out of A, B, and Z
B - right handed
histomes
very basic, positive, many amino acids, attract DNA’s negatively charged phosphate groups
5 different histomes
H1, H2A, H2B, H3, H4
3 Types of DNA: A, B, Z
- A = right handed, wider, thicker, shorter
- B = right handed, most common
- Z = left handed, weird and long
nucleoside
- base + sugar, no phosphate group
- adenosine, guanosine, cytidine, uridine, thymidine
structure of nucleotide
- bases attach to the sugar’s 1st carbon
- phosphate attaches to 5th carbon
- next base attaches to 3rd carbon
- forms ester bonds
- phosphodiester bonding between 5’ carbon - phosphate - 3’ carbon
hydrogen bonds connect bases
- A:T = 2 bonds, easier to break
- C:G = 3 bonds, harder to break
Linus Pauling
worked with proteins, proposed a-helix primary structure using ball and stick models
Franklin + Wilkins
- used x-ray diffraction to study wet DNA fibers, determine structures
- analyzed crystals of molecules using diffraction pattern
- photo 51 super famous
- key discoveries: DNA must be helical, more than 1 strand, 10 bp per turn
Erwin Chagraff
- pioneered DNA isolation tech
- took DNA of over 200 different specimens and discovered that genomes are generally similar, also [A] = [T] and [C] = [G]
- Chargraff’s rule - concentrations are equal
- evidence for multiple strands theory
DNA specifics
- 10 bps, 3.4 nm per turn
- 2 nm width
- two antiparallel strands
- right handed (clockwise) spiral
- major/minor grooves determine what proteins can be expressed
- only certain proteins can bind and interact with certain bases
- bases oriented with flat parts facing each other
- only 1 DNA strand used in RNA synthesis
length of DNA strands
100-1000 nucleotides
RNA is similar to DNA except…
- uracil
- ribose has 2’ OH
- lots of types
Eukaryotic ribosome
80s (40s and 60s subunits)
more complex
Prokaryotic ribosome
70s (50s and 30s subunits)
RNA can form…
short double-stranded sections with double helix (secondary structure) ex. stem and loop structures
DNA polymerases in prokaryotes
5
Meselson + Stahl
able to distinguish between part and daughter strands using 15N and 14N (light and heavy nitrogen)
determined DNA replication to be semi-conservative
what does DNA replication look like in prokaryotes?
replication goes around the circle in both directions and ends at terminus region
(easier in prokaryotes than eukaryotes)
nucleases
destroy nucleic acids
dimeric DNA polymerase
2 DNA poly IIIs work at the same time to replicate leading and lagging strands, move as a unit
- continuous synthesis of leading strand
- discontinuous synthesis of lagging strand
number of DnaA boxes in e. coli
usually 5
how many origins of rep. per prokaryotic chromosome
1
ori C
origin of replication in E. coli
DnaA boxes
- DnaA protein starts replication process by binding to boxes, containing specific sequences
- rich in T and A
- sometimes called 9mers (have 9 bps)
- oligomerization domain allows DnaA to bind to each other
- DNA binding domain allows DnaA to find boxes
AT-rich regions
- sites where DNA strands separate (bonds are weaker here, fewer H bonds)
- also called 13mers
GATC methylation sites
- help regulate DNA replication
- region gets methylated when A gains methyl group on both sides prior to replication
- 11 per sequence
hemimethylated DNA
- when only 1 strand has a methyl group
- SeqA binds to hemimethylated DNA
DAM
DNA adenine methylase
DnaA proteins
- bind to DnaA boxes and each other
- additional proteins bind and cause DNA to bend
- strands separate at AT-rich region
Dna B/helicase
(6 subunits) bonds to origin, unwinds DNA
1. travels in 5’→3’ direction, uses energy from ATP
DnaC
escorts DnaB/helicase to the replication site, drops it off and leaves 🚕
SeqA proteins
- bind to hemimethylated proteins to prevent DNA replication from starting again too early
- keeps DnaA away from boxes
- maintains correct number of chromosomes
DNA B/helicase in unwinding
breaks down hydrogen bonds of 2 strands
topoisomerase (DNA gyrase)
travels ahead and relieves supercoiling caused by unwinding
SSBPS (single-stranded binding proteins)
bind to separated DNA strands to keep them apart
- otherwise, exposed bases could H-bond with individual nucleotides
termination sequences (ter sequences)
opposite the ori C in E. coli
- T1 stops counterclockwise forks
- T2 stops clockwise forks
tus protein (termination utilization sequence)
binds to ter sequences to stop movement of replication forks
what starts dna synthesis?
RNA primers
RNA primers
- created by 🧬DNA primase
- usually 10-12 nucleotides long
- leading strand has 1 primer, lagging strand has multiple
- eventually removed + replaced w/ DNA
DNA poly I
excises RNA primers, replaces w/ DNA
- 1 polypeptide
- 5’ to 3’ exonuclease activity - removes primers
- 5’ to 3’ polymerization activity - puts in DNA
- 3’ to 5’ exonuclease (proofreading) activity
- stops and goes back to fix mistakes - first line of defense against errors
DNA poly III
synthesizes new strand of DNA to fill in gaps
- 5’ to 3’ polymerization activity
- does most DNA replication
- 3’ to 5’ exonuclease activity fixes mismatches - does NOT remove primers
- 10 subunits make up DNA poly III holoenzyme
lagging strand synthesis
- lagging strand is looped, allowing DNA poly III to synthesize in the normal 5’ to 3’ direction
- poly is moving towards rep. fork
- clamp releases the lagging strand after completing each Okazaki fragment
- clamp loader complex reloads the polymerase at the next RNA primer, forms another loop
- process repeats
DNA ligase
links together Okazaki fragments of lagging strands
3 subunits of DNA poly III core
alpha
epsilon
theta
dna poly III alpha subunit
synthesizes DNA by catalyzing bonds between adjacent nucleotides
dna poly III epsilon subunit
proofreading DNA ability
dna poly III theta subunit
stimulates DNA proofreading ability
gamma complex
gamma subunits
delta and delta prime subunits
chi and psi subunits
gamma subunits
load the 🧬beta clamp to attach the enzyme complex to DNA
delta and delta prime subunits
load and open the beta clamp to attach it to DNA
chi subunit
supervises single-stranded binding proteins (which hold DNA open during replication)
psi subunit
interacts w/ gamma and chi subunits
beta clamp
attaches to DNA to allow DNA polymerase to slide along w/o falling off
Tau subunit
dimerization of poly III core complex allows holoenzymes to interact w/ both strands at once
primosome
helicase + primase, interact during replication
replisome
helicase + primase + poly III
replisome is made up of
dimerized DNA poly III, primosome
5 eukaryotic DNA polymerases
α, β, γ, δ, ε
eukaryotic DNA poly α
synthesizes RNA primer, initiates DNA synthesis and lagging strand
β clamp
forms dimer in ring shape around DNA, keeps DNA poly III on DNA strand for longer - can polymerize at least 500,000 nucleotides at a rate of 750 nucleotides/second
γ subunit
replicates mitochondrial DNA
δ DNA poly in Eukaryotes
synthesizes leading strand, fills in gaps after primer is removed
ε DNA polyamerase
repairs DNA - 3’ to 5’ exonuclease
eukaryotic DNA replication
- large linear chromosomes w/ multiple origins of replication
- chromatin is packed tightly in nucleosomes
- many rep. bubbles
end replication problem
how does DNA replication end in eukaryotes when DNA poly I cannot easily remove primers and fill in gaps?
- gaps at the end need an OH group to end replication
- DNA poly III can’t fix it from scratch
- cells will destroy single stranded unfinished DNA
telomeres
- put in place by 🧬telomerase
- made up of RNA and proteins
- the aglets of DNA strands!
- telomeres provide stability, protect ends of chromosomes from nucleases, protect chromosomes from end-to-end fusion
- when they become too short, they stop allowing replication - cell stops dividing and dies
senescence
when cells stop dividing and die
telomeres sequence
usually TTAGGG
- RNA is complementary -AAUCCC
telomerase
- ribonucleoprotein - the protein is a reverse transcriptase
- most active in germ cells which constantly regenerate, and stem cells
- somatic cells lack telomerase activity
enzymatic activity of telomerase
- binding to 3’ overhang region of chromosome
- polymerization - synthesizes 6-nucleotide repeating sequence
- translocation - moves 6 nucleotides to the right
promoter
specific DNA region designed to start transcription
+1
transcriptional start site
direction of transcription
3’ -> 5’
upstream towards 5’ end of coding strand
denoted by negative #s
-10 and -35 consensus sequences
- recognized by RNA poly structure
- TTGACAT and TATAAT (Pribnow box)
Pribnow box
TATAAT
RNA polymerase holoenzyme contains
core and sigma factor
RNA poly core
- 2 alpha subunits
- 1 beta subunit
- 1 beta’ subunit
- 1 omega subunit
sigma factor
assigns specificity to RNA poly
- bonds to the -10 and -35 regions of promoter so transcription starts correctly
- only bonds under certain conditions for gene expression
sigma factors in E. coli
- multiple sigma factors to recognize a distinct set of promoters
- σ70 recognizes housekeeping genes - basic survival, metabolic function, etc.
- σ32 recognizes promoters for genes needed to survive under stressful conditions like high temperatures - leads to production of heat shock proteins
- σ38 recognizes promotors for when cells are starving and in a state of no growth
DNA-RNA hybrid
- found where the RNA follows leading strand template
- hybrid gets cut off to disconnect mRNA
stages of transcription
initiation, elongation, termination
transcription initiation (prokaryotic)
RNA poly binds to promoter
1. helicase “melts” the two strands apart to form transcription bubble
2. continues until sigma factor falls off (is no longer needed)
elongation
RNA poly unwinds DNA ahead of it, then rewinds it after transcription
elongation factor
replaces sigma factor
ex. NusA
termination
strands ditch DNA poly
2 possible methods - rho-dependent or intrinsic
rho-dependent termination
- depends on transcription terminator protein with both ATPase and helicase activity, accounts for 20%-50% of termination events
- this protein has 6 subunits
- binds to rut (Rho utilization site)
- rich in cytosines
- 70 nucleotides of growing RNA chain will wrap around the Rho protein, activate ATPase activity
- uses energy released to translocate up to the DNA-RNA hybrid - speeds up, sites ahead slow down RNA poly so Rho can catch up
- RNA poly pauses at Rho sensitive pause sites - stem-loops like roadblocks
- at hybrid region, Rho uses helicase activity to separate and release RNA molecule - RNA poly falls off
intrinsic termination
rho-independent, relies on a signal within RNA to terminate
1. hairpin of RNA strand forms when RNA is released from hybrid → stem binds to NusA → A and U strands have weak bonds, causes overall destabilization → mRNA and RNA poly fall off → transcription ends
_____ and _____ take place in eukaryotic nucleus at the same time
transcription; RNA processing
steps of RNA processing
- addition of a modified guanosine cap at the 5’ end
- remove introns from premessenger RNA
- combine exons
- polyadenylation (cleavage) at 3’ end
how many RNA poly in eukaryotes
3 - each transcribe different types of genes
E RNA poly I
rRNA in nucleolus
E RNA poly II
mRNA, small nuclear RNA in nucleoplasm
E RNA poly III
5S RNA, tRNA in nucleoplasm
eukaryotic promoters are first recognized by…
General Transcription Factors (GTFS) a.k.a. basal transcription factors
GTFs - examples
TFIIA, TFIIB, TFIID, TBP, TFIIE, TFIIF, TFIIH
important GTFs
TFIID and TBP (TATA Box Binding Protein) start nucleation process
GTFs function
attract RNA poly II to start at transcription site
preinitiation complex (PIC)
GTFs + RNA II poly core
RTFs
regulatory transcription factors - bind to specific sequences
transcription elongation
occurs inside transcription bubble - capping, phosphorylization of CTD, phosphates removed, splicing
capping
nascent RNA emerges from RNA poly II, cap is added to 5’ end by proteins that interact with CTD
- cap made up of 7-methylguanosine residue linked to transcript by 3 phosphate groups
- protects RNA from degradation
- required for translation
what is the cap made up of
7-methylguanosine residue
CTD
- carboxy terminal domain - gets phosphorylated - recruits phosphorylation enzymes (🧬tinases)
- controls all processing
phosphotases
new enzymes brought in in elongation- remove phosphates after capping
splicing
removal of introns, joining of exons, done by the 🧬spliceosome
1. introns almost always have GU at the 5’ end and AG at the 3’ end - GU-AG rule
spliceosome made up of
SNURPS: small nuclear RNAs and ribonucleoproteins
SNURPS examples
U1, U2, U4, U5, U6 (U3 is chilling in the nucleolus)
sequences that end RNA elongation
AAUAAA or AUUAAA
end of elongation
- enzyme recognizes sequence, cuts off the end of the RNA ~20 bases further down
-adds poly-A tail - sequence of 150-200 adenine bases
