-
repetitive process of replication, transcription, and translation
build polymers of nucleotides or amino acids
-
3 major steps of replication, transcription, and translation (RTT)
- initiation
- elongation
- termination
-
when RNA polymerase binds to promoter, it forms a
closed complex
-
RNA polymerases binds promoter, 10 and 35 bases upstream of start site
-
step in RTT, binds polymerizing machine, first monomer to template
initiation
-
3 structures involved in initiation
- DNA polymerase
- RNA polymerase
- ribosomes
-
step in RTT that reads template and adds next monomer
elongation
-
3 structures involved in elongation
-
step in RTT that release machine and complete product
termination
-
RNA polymerase is a large molecular machine
-
this enzyme has 4 protein units in one complex
RNA polymerase
-
function of RNA polymerase
binds DNA and reads sequence
-
Where does RNA polymerase bind to DNA
sigma factor
-
3 functions of sigma factor
- specificity
- guides RNA polymerase to DNA
- promoter
-
-
2 characters of sigma factors
- each for subset of genes
- contain diff promoters
-
function of RpoD sigma70 genes
housekeeping
-
most genes from sigma factors are
RpoD sigma 70
-
RpoD, sigma 32 functions when
activate when the cell is stressed by heat
-
heat shock response proteins
-
sigma factors binds core RNA polymerase which forms
RNA polymerase holoenzyme
-
mRNA func
encodes protein
-
rRNA func
synthesizes protein
-
tRNA func
shuttles amino acid
-
sRNA
controls transcript and lation
-
tmRNA func
free ribo stuck on damaged mRNA
-
catalytic RNA func
carries out enzymatic rxns
-
genetic code consists of nucleotide triplets
codons
-
64 possible codons
61 specify AA (w/ start codon)
3 stop codons
-
genetic codes are degenerate or redundant
-
multiple codons can encode same amino acid
-
given DNA sequence in transcript and lation can encode 2 mRNAs
-
in protein synthesis, transcript and lation are directional and one is used as a template
-
in protein synthesis, a set of tRNAs bind individual amino acids
-
2 characters of tRNA
- have specific shape
- have 3 base anticodon (base pair to codons in mRNA)
-
proteins add AA to tRNA, enzyme used is
aminoacyl-tRNA transferase
-
when proteins add AA to tRNA, what kind of E is required and what does it get reduced to
-
function of aminoacyl tRNA synthetases
enzyme that matches amino acid to correct tRNA
-
DNA and proteins can be labeled separately by using radioactive S and P, which letter goes to which
-
2 types of gene transfer
- vertical
- horizontal transmission
-
vertical transmission is the transfer from
parent to child
-
horizontal transmission is the transfer of
small pieces of DNA from one cell to another
-
produces a functional RNA which usually encodes a protein
structural gene
-
regulates the expression of structural gene
DNA control sequence
-
DNA control seq does not encode an RNA
-
dna polymer is a polymer made up of nucleotides
-
each nucleotide of DNA is made up of 3 parts
- nitrogenous bases
- deoxyribose sugar
- phosphate
-
the 2 nitrogenous bases types
-
-
-
nucleotides are connected to each other by
5-3' phosphodiester bonds
-
4 components to the nucleic acid structure
- sugar (deoxyribose)
- 3 phosphate groups on 5' C
- phosphate links to 3' OH of the next base
- base of DNA attached to sugar
-
order of bases on
DNA
RNA
- Adenine, guanine, cytosine, thymine
- adenine, guanine, cytosine, uracil
-
3 ways RNA is different from DNA
- single stranded
- contains ribose sugar
- uracil replaces thymine
-
complimentary sequence of
5'-ATG-CTT-AAG-3'
5'-CTT-AAG-CAT-3'
-
DNA is packed to fit the cell
-
ex of how DNA is packed to fit the cell
nucleoid of e.coli is composed of circles of dsDNA 1500x the size of the cell
-
how is nucleoid of E.coli able to be packed into the cell 2
- multiple loops are held by anchoring proteins
- each look has coiled DNA
-
unsupercoiled DNA=
1 wind for 10 BP
-
-
supercoiling compacts DNA
-
this kind of winding is more frequently, overwinding occurs
positive supercoiling
-
this kind of winding has winding that is less frequently, underwinding occurs
negative supercoils
-
this kind of coiling twists DNA
supercoils
-
what helps to regulate supercoils
topoisomerases
-
3 kinds of topoisomerases
-
this kind of topo relieves torsional stress caused by supercoils
type I
-
this kind of topo has DNA gyrase and introduce negative supercoils
type II
-
this kind of topo reverse DNA gyrase and introduce positive supercoils
archael
-
DNA replication
(semiconservative replication)
copies info from one strand to a new complimentary strand
-
why is DNA known as semiconservative replication
due to accuracy check
-
how to DNA replication start off 2
- melt double stranded DNA
- polymerize new strand complementary to each melted single strand
-
6 major proteins involved in DNA replication
- DNA-A
- DNA-B
- DNA primase
- DNA pol III
- DNA pol I
- DNA gyrase
-
DNA-A function
initiator protein
-
-
DNA primase function
synthesis of DNA primer
-
DNA pol III function
major replication enzyme
-
DNA pol I function
replaces RNA primer with DNA
-
DNA gyrase function
relives DNA supercoiling
-
where does replication begin
at oriC
-
When DNA melts at oriC, what 2 things are occuring
- DNA-A activates
- Seq-A represses
-
when replication begins, what 2 things are happening
- DNA melts at oriC
- polymerization follows melting around the chromosomes
-
What helps to melt DNA for replication
DNA helicase
-
How does helicase melt DNA 2
- loader places helicases at each end of the origin
- one helicase moves in each direction to copy each genome
-
when helicase recruits primase, what 2 things occur
- primase will begin the replication
- RNA primer forms 3' OH for DNA to attach
-
first cells were thought to use RNA not DNA
-
when primer recruits clamp loader to each strand, what happens
clamp binds DNA polymerase III to strand
-
polymerase replicates from 5 -->3 on each strand
-
energy for polymerization for DNA replication comes from where
phosphate groups on added bases
-
when polymerization occurs, it must add a new base to 3 OH of a chain, while new nucleic acid grow to extend 3 end
-
each replication fork has two strands
-
at each fork in replication there is a steadygrowth of new leading strand (follows helicase)
and a single DNA strand for half of chromosomes
-
2 occurs while the lagging strand grows during replication
- polymerase continues to the previous primer
- clamp loader places primase on new site
-
when the lagging strand grows, it is done by the okazaki fragment
DNA has 1000 present in the okazaki fragments
-
RNAse H function
removes primers
-
there is 1 primer for each leading strand in DNA rep
There are many primers of lagging strand in DNA rep
-
there is one primer per okazaki fragment
-
the gaps of the DNA rep are filled by ____ and the nicks are sealed by _____
-
when both forks move to ter sites 3 things occur
- movement is simulatenous
- move opp ways until they meet again at terminus
- replisomes are actually stationary
-
DNA gets threaded through replisomes
-
are extrachromosomal pieces of DNA
plamsids
-
low copy number plasmids 2
- 1 or 2 copies per cell
- segregate similarly to chromosome
-
high copy number plasmids 3
- up to 50 copies per cell
- divide continuously
- random segregation to daughter cells
-
2 ways plasmid replication can occur
- bidirectional replication
- unidirectional replication
-
plasmid bidirectional replication is similar to chromosomal replication
-
this type of plasmid replication is used by many bacteriophage viruses
unidirectional
-
how unidirectional plasmid replication work
- starts at nick bound by RepA protein
- provides 3 OH for rep
- helicase moves around plasmid repeatedly
- complementary strand synthesized
-
plasmid genes are advantageous under special conditions
-
plasmid genes can perform 5 functions amongst others examples
- antibiotic resistance
- resistance to toxic metals
- metabolize rare food sources
- virulence genes to allow pathogens
- allow symbiosis
-
2 ways DNA can be manipulated
- restriction enzymes cut DNA at specific sites
- PCR uses short oligonucleotides
-
restriction enzymes are used to
normally protect bacteria from viral DNA
-
when the restriction enzyme cuts the DNA, it reveals location of specific sequence
-
PCR use of short oligonucleotides cause these 3 to occur
- primers binding to complementary sites
- archael enzymes syn DNA
- amplifies segment of DNA
-
restriction endonucleases
cleaves DNA at specific recognition sites which are usually 4-6 bp and palindromes
-
when restriction endonucleases occurs, it may generate blunt or staggered ends
-
agarose gel electrophoresis function
can be used to analyze the DNA fragments obtained by treatment with restriction enzymes
-
dna amplification
polymerase chain reaction
-
4 events in PCR
- denaturation
- annealing
- extension
- repetition
-
PCR uses heat stable DNA polymerase
-
whole genome sequence 3 characteristics
- break genome into 1,000's of pieces
- determine sequence of many short pieces
- computer determine sequence overlap to recreate entire genome sequences
-
most commonly used DNA sequencing method relies on the
sanger dideoxy strategy
-
what is the sanger dideoxynucleotide strategy
incorporation of 2',3'-dideoxynucleotide into a growing chain preventing further elongation
-
bacterial chromosome is packed in a series of protein bound loops collectively called
nucleoid
-
are enzymes that super coil DNA
topoisomerases
-
DNA replication is divided into 3 phases
- initiation
- elongation
- termination
-
DNA replication:
initiation occurs
elongation occurs
termination occurs
- at the origin (oriC)
- at the replication fork
- at the terminus
-
each phase of replication requires a # of different proteins
-
plasmids are autonomously replicating extrachromosomal DNA elements
-
plasmids benefit the host under certain conditions
-
4 ways DNA can be analyzed
- restriction enzymes
- gel electrophoresis
- PCR
- DNA sequencing
-
polymerase unwinds DNA at promoter known as
open complex
-
once polymerase begins to unwind, what happens to the sigma factor
it is released
-
which part of the RNA polymerase recognizes the -10 region of a promoter
region 2 of sigma factor
-
core polymerase adds RNA to 3' end
-
energy for base addition comes from the base
mRNA (n) + NTP = mRNA (n+1) + diphosphate
-
added base in transcription elongation when polymerase adds RNA is
complementary to template strand
-
mRNA in transcription elongation of RNA has the same sequence as sense strand
-
For transcription termination of RNA is depends on what to help the termination
rho-dependent
-
When termination of RNA transcript occurs, what happens
RNA polymerase slows at pause site (has a GC-rich sequence)
-
how does Rho cause termination of RNA transcription
- Rho factor binds to mRNA
- slides along the mRNA to polymerase
- Breaks off polymerase and mRNA off of DNA
-
3 characteristics of Rho independent termination
- DNA seq containing intrinsic terminators
- inverted repeats followed by runs of Us
- stem loop structure in RNA
-
what 4 subunits are present during the closed complex during transcription
alpha, beta, sigma, gamma
-
transcription and translation are directional
- given DNA seq can encode 2 mRNAs
- usually only one strand is used as template
-
translation of RNA to protein
initiation
initiation factors bind ribosome to start codon
-
translation of RNA to protein
elongation
EF- Tu, EF-G bring GTP energy (polymerization)
-
polymerization
movement of ribosome along mRNA
-
translation of RNA to protein
termination
release factors undock ribosome from mRNA
-
3 characteristics of tRNA protein synthesis
- bind to individual amino acids
- have a specific shape
- have a 3 base anticodon
-
proteins adding amino acid to tRNA is called
- aminoacyl-tRNA transferase
- requires E of ATP--> AMP
-
wobble position of anticodon during protein synthesis of tRNA allows
certain tRNAs to read more than 1 codon
-
aminoacyl-tRNA synthetases function
an enzyme that matches amino acid to correct tRNA
-
protein polymerase
ribosome
-
structure of ribosome 3
- 52 proteins
- 3rRNAs
- 2 subunits
-
ribosome can bind these 2
-
rDNA operon is transcribed as a single molecule
-
replication of tRNA has multiple copies of the operon
-
peptidyltransferase
23S rRNA of 50S subunit catalyzes peptide bond formation
-
the 70S ribosome harbors 3 binding sites for tRNA
-
This site binds incoming aminoacyl-tRNA
acceptor site
-
this site harbors the tRNA with the growing polypeptide chain
peptidyl tRNA site
-
this site binds a tRNA recently stripped of its poplypep
exit site
-
alignment of bacterial structural gene with its mRNA transcript
-
this antibiotic that affect translation inhibits 70S ribosome formation
streptomycin
-
this antibiotic that affect translation inhibits aminoacyl-tRNA binding to the A site
tetracycline
-
this antibiotic that affect translation inhibits peptidyltransferase
chloramphenicol
-
this antibiotic that affect translation triggers peptidyltransferase prematurely
puromycin
-
this antibiotic that affect translation causes abortive translocation
abortive
-
this antibiotic that affect translation prevents translocation
fusidic acid
-
enzymes modify translated proteins
-
fMet removed from N-terminus in protein modification
-
3 groups added to AA in protein modification
- phosphoryl
- methyl
- adenylate
-
a protein can be cleaved or refolded by helping enzymes in protein modification
-
protein structure is determined by
-
2 kinds of chaperones involved in protein folding
- GroEl-GroES complex
- DNAK protein
-
function of Gro-EL GroES complex
refolds denatured proteins
-
how are proteins refolded
by the use of ATP
-
barrel shaped proteins that are apart of the GroES complex function
helps the pro to be refolded fit into the center
-
all cells constantly rebuild themselves
proteins are degraded when not needed
-
proteins survive for minutes to days
sequence, shape, function determine half life
-
proteases cut proteins
cut at specific amino acid sequences
-
proteasomes degrade proteins and have a barel shape
-
eukaryotes add signal to proteins, what does the tag do
ubiquitin tag causes degradation
-
protein traffic: many bac proteins reside in cytoplasm, others are targeted to other sites, and have signal sequence
-
4 targets proteins can be targeted to
- PM
- periplasm
- gram- outside mem
- secreted outside bacterium
-
signal sequence
target protein for transport
-
3 characteristics of protein secretion: signal sequence
- n-terminal AA
- bound by SRP
- target ribosomes to plasma membrane
-
for signal sequencing, they target ribosomes to plasma membrane. what two ways can the proteins be inserted into their final destination
- some proteins enter plasma mem directly
- others require secYEG translocon
-
post translational secretion: SecB function
protects unfolded polypeptides
-
post translational secretion: SecA function
uses ATP to push peptide through SecYEG
-
post translational secretion: Signal peptide function
cleaves N-terminal sequence
-
post translational secretion: TAT system
exports unfolded proteins using PMF
-
gram- bactria need to export proteins completely out of the cell
-
six elegant secretion types have evolved, labeled type I-VI, what are two of their functions
- some deliver te exported proteins to other dedicated transport proteins in the periplasm
- others provide nonstop service
-
genome provides raw DNA sequence
-
what DNA sequences encode proteins
open reading frames
-
what DNA sequences control expression
promoters
-
what proteins perform specific function
biochemical analysis of protein function
-
genes duplicated via appearance of new species
orthologs
-
genes duplicated within a species
paralogs
-
in orthologs, identical function in diff organism
-
in paralogs, they perform slightly diff tasks in cell and can develop new capabilities
-
transcription is carried out by
a complex enzyme called RNA polymerase
-
function of sigma factor
recognizes the promoter and the core polymerase elongate the RNA strand until a termination signal is reached (may or may not depend on Rho protein)
-
there are 6 diff classes of RNA
-
tRNA shuttle amino acid to ribosomes
-
3 phases of RNA translation
- initiation
- elongation
- termination
-
translation RNA initiation
ribosomal subunits come together
-
translation RNA elongation
AA are polymerized
-
translation RNA termination
completed protein is released
-
cell possess diff protein degrading machines
-
bioinformatics
use of computer resources to mine and compare genome of organisms
-
during DNA replication, each nucleotide adds on to
hydroxyl group on the 3C of the sugar
-
restriction enzymes do not cut bac DNA that produce them because
the DNA is protected by site-specific methylating enzymes
-
if the DAM enzyme is inhibited in E.coli, then the next generation time
will increase due to more persistent seqA binding
-
why is the shuttle vector named so
contains a replication origin compatible with Ecoli and a second origin that will allow the plasmid to replicate in a eukaryote or archae
-
organisms differ in their gene size
-
a gene is
a string of nucleotides that can be used as a template to produce a functional RNA
-
bactria, archae, and eukaryotes all have what kind of genomes
double stranded DNA genomes
-
what is present in DNa sequencing reaction but not in vivo
dideoxynucleotides
-
only RNA contains a hydroxyl group on the 2' carbon of ribsose
-
At 72 degree stage
thermostable DNA poly syn DNA
-
denaturing of DNA refers to
how the helix separates into single strands
-
supercoiling of DNA may be effected by antibiotics
-
example of verticle transmission
cell division
-
the enzyme DNA primase is
RNA polymerase
-
Eukaryotic genomes are composed of non coding DNA while prokaryotes have mostly coding DNA
-
semiconservative nature of DNA rep indicates that
each daughter cell receives one parental strand and one newly synthesized strand
-
most bacterial species DNA is
- supercoiled
-
55 degree stage of PCR
primers anneal to the denatured DNa strands
-
organisms differ in genome size
-
what shows the increasing number of genes
gene, operon, regulon
-
initiation of DNA rep in bacterial species
is influenced by environmental factors
-
X replication differs from rolling circle method of plasmid rep in that
only X rep produces okazaki fragments
-
a plasmid singly cut with a restriction enzymes that leaves cohesive ends
can ligate to any DNA cut with the same restriction enzyme
-
bacterial genome consists of
the genome structure depends on species
-
an example of DNA control sequence
promoter region of a gene
-
the plasmid encoded ParR and ParM proteins are involved in
plasmid partitioning
-
enzymes that regulate DNA supercoiling are called
topo
-
95 degree strand in PCR
dna strands denature
-
E.Coli DNA pol III has what activity
3-5' exonuclease
-
this gene usually produces an RNA molecule that encodes a protein
structural gene
-
this regulates the expression of a structural gene
DNA control sequences
-
binding sites for regulatory proteins can do these 2
activate or inactivate that promoter
-
prokaryotic genomes are made up of
plasmids are made of
-
functional units of DNA sequences include these 2
- structural gene
- regulatory sequences
-
prokaryotic and plasmids can be circular or linear
-
prokaryotes have large amounts of coding DNA while eukaryotes have small amounts of coding DNa and large amounts of non coding DNA
-
noncoding regions of DNA in eukaryotes is composed of these 2 and their functions
- enhancer sequences, needed to drive transcription of eukary promoters
- DNA expanses that separate enhancers
-
can function at large distances from the gene they regulate
enhancers
-
is the DNA seq immediately in front of a gene that is needed to activate the genes expression
promoter
-
how are all the genes in an operon situated and controlled
- head to tail on the X
- by a single reg sequence located in the front of the first gene
-
the single RNA molecule produced from the operon contains all the
info from all the genes in the operon
-
a collection of genes and operons at multiple positions on the X can hold membership in a
regulon
-
horizontal gene transfer mechanism requiring cell to cell contact can transfer large segments of bac X
conjugation
-
transfer of X is non specific and requires recomb and sometimes it might take different amounts of time for it to occur
-
adenine and thymine
cytosine and guanine
# of hydrogen bonds
-
DNA with higher amounts of CG require higher denaturing temperatures due to it having 3 hydro bonds
-
denaturing is much faster than renaturing since renaturing is random with a hit or miss type ordeal
-
this is necessary step in decoding of genes to make proteins
hybridization
-
what causes the X of E.coli to have a - charge of the cyto
all the phosphates in the backbone are unprotected and negatively charged
-
bacteria nucleoid is distributed throughout the cyto instead if being compacted like the eukary
-
boundaries of supercoiled loops are defined by anchoring proteins called
histones
-
supercoiling of one domain of DNA is maintained independently of other loops
-
torsional stress of overwinding is relived how
when DNA twists upon itself causing + supercoiling
-
how are - supercoils formed
if one end of DNA molecule is turned in the same direction as the helix, underwinding DNA
-
- supercoiling is easier to separate because DNA is underwound
-
+ supercoiling DNA is harder to denature because
it takes excess E to separate overwound DNA
-
description of how spatial features of an object are connected to each other
topology
-
supercoiling changes topology of DNA
-
enzymes that change DNA supercoiling are
topoisomerases
-
to maintain proper DNA supercoiling levels what must a cell do
balance the activities of 2 types of topoisomerases
-
this type of topo are usually single proteins
have multiple subunits
-
smallest genome known for free living microbes encodes
480 proteins possible
-
hydrogen bonding and interactions between the stacked bases hold together complimentary strands of DNA
-
bacteria, eukary and most archae have - supercoiling
-
these topo cleave 1 strand of a DNA molecule to relieve supercoil
type 1
-
this topo cleaves 2 strand of DNA and use ATP to introduce supercoils
type 2
-
circular microbial X replicates
bidrectionally
-
how is initiation of replication determined
- DNA methylation
- binding of specific initiator protein to the origin seq
-
3 things that can cause replication to begin again
- origin is fully methylated
- seqA dissociates
- DNAA-ATP conc rises
-
main replication pol known also as a molecular macine
polIII
-
function of exonuclease activity of polIII
- cleaves the phosphodiester bond releasing improperly paired base from a growing chain
- (once mispaired is fixed, polIII continues)
-
polI enzyme enters after the rnase and syn the DNa patch using he 3OH end of the preexisting DNA fragment as a priming site
-
template DNA is threaded through the replisome, the helicase pulls apart the 2 strands of the DNA helix, which causes
DNA ahead of the fork to twist introducing + supercoiling
|
|
