Exam 3
Chapter 9
DNA strands
Anti parallel
10 bases per turn
Nitrogenous bases face each other in order to hydrogen bond them together
3.4 nm per turn (.34 nm per base)
What are the different ways in which the structure of DNA is finalized?
Scientists
Linus Pauling
Maurice Wilkins/ Rosalind Franklin
Erwin Chargaff
Would purify and wet DNA
Found two common structures seen
Alpha helical
Beta Region
Both involve hydrogen bonds
Used ball and stick to find structure
Expose to X-Rays
Refraction pattern verified the double stranded and helically coiled rather than single stranded
10 bp per turn
More interested in the role played by the nitrogenous bases (a,t,c,g)
Took cells from species of different organisms
Exposed to detergent to promote all unwanted components and isolate chromosome
Remove histones= treat with protease
Just DNA
Expose to acid to hydrolyze and isolate into individual nitrogenous bases
Analyzed with spectrophotometer
A&T proportional G&T proportional
Chargaff's rule
Let us understand the capability of replication through unwinding and making copies
There is a lot of Mg inside of the nucleus so may assumed that Mg had much to do with the structure of the DNA
Mg++ crosslinks
Watson and Crick
Incorrect hypothesis
Double Helix model
Sugar/ Phosphate on outside in contact with water
Face inward
Purines
Pyrimidines
Two carbon
One carbon
Thymine and cytosine
Adenine and Guanine
Three forms of DNA
BDNA
ADNA
ZDNA
Most stable
Right handed coiled double helix
10 bp per turn but actually 10.5 technically
Nitrogenous bases arranged perpendicular across a central axis
Also Right handed
Much thinner, no distinctive grooves
Grooves
Space filling model
Every atom considered to be a sphere
So each time a right handed turn takes place there is a physical groove that forms
Proteins targeting binding sites typically target major groove
Not seen often but in low humidity
When bacteria are exposed to hazardous conditions they make endospore capable of withstanding harm created by UV radiation
Accomplished by taking on A form
Occurs with many terminal repeats (transposable elements) (moderately repetitive and highly repetitive
Occurs when there are many Cs in a sequence especially
Left handed
12 bases per turn
Phosphates have zig zag orientation
brought closer together
causes them to repel each other
Often seen at transcription sites
When DNa starts replicating the first strand seen is a Dna RNA hybrid
DNA can also form a triple helix
Enzymes in yeast have capacity to bind to triplex DNA
We can artificially induce third strand of DNA to bind to major strand
This stops replication, transcription, and recombination
DNA is not naked in eukaryotes but associated with histones
Ribonucleic acid
Nucleotides still the building blocks
Phosphodiester connects nucleotides
Different than dioxynucleotide because of sugar
in ribose 2nd carbon has the hydroxyl group and a nitrogenous base added to the first carbon
Stem loops regulate gene expression
How do bulge or internal loops occur
RNA have complimentary regions interspersed w/ non complimental regions you will have several types of loops formed
Loops form where there is absolutely non complimentary
Chapter 11
Replication requires unwinding
Three models of replication
Conservative model
Semiconservative model
Dispersive model
Both parental strands of DNA remain together following DNA replication
Original arrangement is conserved and daughter strands remain together
Double stranded DNA is half conserved following the replication process
Newly made DNA contains one parental strand and one daughter strand
Mix of parental and daughter DNA within one strand
To prove separated by density
N15 represented parental while N14 represented daughter
First gen showed half heavy so conservative and semiconservative were possible
Second gen showed half heavy and half light which is only consistent with semi conservative
Bacterial DNA replication
When two replication forks meet then replication is done, proceeds bilaterally
Intertwining circles are possible while replicating circular DNA ?????
Topoisomerase
Capable of doing double stranded break and therefore can relieve
Initiation of replication (FIRST PART)
Three sequences within OriC region
DnaA boxes
AT Rich region
GATC methylation sites
Every adenine in GATC site must be methylated
Causes the two strands of DNA to separate
Also need DnaA proteins
- Bind to DnaA boxes in the form of 20-40 DnaA ATP complexes
- DnaA wraps around protein and tension causes first break in two strands
This break is further enhanced by histone like proteins and integration host factors
ATP is necessary to supply energy
- After first break is formed Helicase binds
Helicase is a heximer (six identical subunits)
DNA passes through hole
two helicases bind, one to each strand
DnaA boxes help the helicase to come and bind
Helicase-DnaB
THIS SHIT IS NOT RELATED AT ALL TO ADNA DO NOT MIX THIS UP YOU FUCKING DUMBASS PIECE OF SHIT FUCK YOU
DnaC also helps the DnaB (helicase) to bind (aids the DnaA)
Methylation of cytosine is silencing transcription while methylation of adenine is beginning replication
Break occurs in the AT rich region
There are 5 DnaA boxes
- Helicase continues the breaking of Hydrogen bonds
Energy from ATP hydrolysis is used
Establishes the replication fork
Once established replication will proceed bidirectionally
- Single strand binding protein binds to Separated DNA strands to keep them apart
DNA has innate ability to form hydrogen bonds back
polar compounds face each other
As helicase breaks the strands apart positive super coiling develops topoisomerase II? ahead of the unwinding (the opposite direction)
- (sort of simultaneous) must be applied to relieve positive supercoiling
DNA synthesis (SECOND PART)
Polymerase
Two drawbacks
Cannot initiate synthesis
Can only catalyze the addition of nucleotides in 5' to 3' direction
incoming nucleotides can only be attaches to 3rd carbon of pre-existing sugar
How many polymerases are present in bacteria
Eukaryotes have 12
Prokaryotes have 5 distinguished by roman numerals
we only look at 1 & 3
2,4,5 are involved in repair
DNA polymerase 1
is only made of one subunit
Only tertiary structure
DNA polymerase 3
Made of several protein units
Therefore also has quaternary structure
Alpha subunit
Beta subunit
Epsilon subunit
Processivity
(continuously adding nucleotides without falling off)
Clamp protein (ring that holds polymerase so it can walk along DNA)
Catalyzes the formation of the phosphodiester bond between the nucleotides
Proofreading
Because DNA polymerase cannot initiate synthesis DNA primase comes in first
similar to RNA polymerase
Makes tiny strand (10-12 RNA nucleotides)
These are called RNA primer
Dna polymerase 3 continues synthesis in 3' direction
DNA polymerase 1 will remove RNA primer and substitute with DNA nucleotides
As this substitution is done, one phosphodiester bond will be missing between strand made by DNA polymerase 1 & 3
This ester bond will be catalyzed by an enzyme called DNA ligase
If a strand is synthesized in the 5' to 3' direction the template is 3' to 5'
Strand types
Leading strand
One RNA primer is made at the origin, DNA pol III attaches nucleotides in a 5' to 3' direction as it slides toward the opening at the replication fork
By looping template parent strand strand in small segments you can reverse direction of strand
Each fragment formed with each fragment formed with each loop is 1000-2000 bp loop
Nucleotides come in the form of triphosphate
For example: deoxyadenosin triphosphate, two phosphates will be released because of water in cell (hydrolysis)
Monophosphate will form ester bond
This bond break actually provides the energy
Lagging strand
Template loop in the form of Okazaki fragments
Occurs away from replication fork
This strand will have hyperactive DNA ligase and primase
Repetitive sequences in bacteria are those sequences necessary for transcription, replication, translation, etc.
There is very little time lag between leading strand and lagging strand
Eukaryotes
prokaryotes
DNA ligase gets energy from ATP hydrolysis
NAD+ breakdown gives energy
What if there is a mutation in the Beta subunit
Other subunits have backup units to fulfill functions but beta does not rate is reduced severely (20 nucleotides per second versus 750
Termination of replication (THIRD PART)
Takes place at a point opposite of the ORiC sequence
Replication goes both clock and counterclockwise
When replication forks meet it is region opposite to where it started
Replication ends because everything is replicated and no template remains
Intertwined circles released by topoisomerase II as topoisomerase I can only perform single strand break
TER sequence
There are two ter sequences on double stranded DNA. One strand complementary to the other
TER sequences on own do not stop replication
Must be bound by protein called termination utilizing substance (TUS)
Only one TUS needed for two ters sequences
Any particular Tus protein will either allow for counter or clock fork to continue but never both as they would overlap
Multienzyme complexes
Primosome
Replisome
Helicase and primase
DNA polymerase III holoenzyme
First enzymes to come into a non-covalent association
associated with the primosome
Proofreading
multiple systems in place
instability of mismatched base pairs
A&T 2H bonds
C&G 3H bonds
Due to instability they will not form ester bond in the opposite strand
Configuration of polymerase
Error rate 1 per 1000 nucleotides
Alpha subunit which catalyzes the formation of phosphodiester bonds
If the DNA strand is distorted it will not fit in the domain of the alpha subunit and will not allow phosphodiester bond to form
This reduces the error rate to 1/100,000 to 1 million
Bacterial DNA multiply rapidly
one replication takes 20-200 minutes (30-40 minutes average)
How is this regulated?
Methylated strand of GATC is parent while non methylated is daughter strand
together they make hemi methylated DNA
When bacteria has grown and is ready to replicate enzyme called DNA adenine methyl transferase converts hemi methylated strand to fully methylated strand
This is the control
How did we discover polymerases other than I?
Mutant analysis
The question was why does DNA replication still occur even with mutated DNA polymerase I
Experiment:
Expose bacteria to mutagenic agent
Put on AGAR plate
Take round velveteen sheet and touch with colonies
One in permissive
One in non permissive
- Identify defective proteins in those which failed to grow
- map mutations along e. coli chromosome
- Provide important starting points
Slow stop mutants
Rapid stop mutants
mutations in initiation process
mutations in replication process
Eukaryotic Replication
Origins of replication
Begins with formation of 14 subunit complex called the pre replication complex (preRC)
Each is about 150-200 bp long in bacteria
In Eukaryotes each is about 50 bp
ORiC
ARS (Autonomously replicating sequence
Three or four copies of the same sequence (many As and Ts)
Similar to bacterial A boxes
The first part of the preRC formed is the ORC (origin recognition complex)
Present from end of mitosis until G1 phase
getting ready to assemble onto ARS
14 proteins
6 member protein
Combines with ATP and allows for helicase attachment
Similar to DnaA ATP complex
Cell division control protein
Promotes the binding of
Homolog 6 or protein 6 homolog (also arrives with ATP)
CDT 1
Controls chromosome licensing and DNA replication
MCM helicase (complex of 6 proteins)
All of these proteins assembled by end of G1 phase
Then DNA replication licensing occurs
Ensures entire stretch of protein replicated once and only once
MCM helicase is bound at origin of replication allowing formation of two replication forks
s phase
Then Firing occurs
This occurs during the G1 phase
Cyclin dependent kinase binds with cyclin and causes moving and functioning of cell
Causes preRC to be converted to an active replication site via phosphorylation (thereby hydrolyzing ATP)
CDC6, CDT1, ORC fall off
MCM helicase moves in the 3'->5' direction eventually falling off
DNA replication proceeds bilaterally from the origin
Polymerases
Normal replication in prokaryotes involves 2 polymerases (1 &3)
Normal replication in eukaryotes involves four
(over a dozen overall)
Alpha
(five overall)
Delta
Epsilon
Gamma
Mitochondrial
All 3 nuclear
Replication protein A
Keeps strands separated
Just like single strand protein in prokaryotes
Synthesis process
Prokaryotes just have DNA primase responsible for synthesizing the first strand of DNA
Eukaryotes also have DNA primase
However, it is present in complex with DNA alpha polymerase
Always synthesizes 10 bp RNA strand followed by a 20-30 bp strand of dna (overall 30-40 bp long strand)
Alpha polymerase will then detach and be replaced by either a delta or an epsilon polymerase
This is known as a polymerase switch
Epsilon polymerase unit has 3'-> 5' template (leading)
Delta polymerase has 5'->3' template (lagging)
Prokaryotes do not have this type of differentiation
In eulkaryotes okazaki fragments are 100-200 bp
In prokaryotes okazaki fragments are 1200 bp
Beta subunit of DNA polymerase III in prokaryotes was responsible for processivity
Eukaryotes do not have that
DNA polymerase I in proks removes RNA primer
DNA ligase catalyzes phosphodiester bond in prokaryotes (NAD+ source of energy)
Processivity in eukaryotes
proliferating in Euks= Proliferating cell nuclear antigen (PCNA)
Ligase still functions in Eukaryotes
Catalyzes the formation of ester bonds in strands synthesized by alpha epsilon or delta DNA polymerase
Eukaryotes use ATP rather than NAD+
As delta DNA polymerase adds nucleotides to the lagging strand it will eventually encounter the RNA primer and keep synthesizing nucleotides, pushing the RNA primer
Pushing will cause a flap
10 bp long
Flap endonuclease has a site that will bind and break down the flap
However the delta polymerase can push DNA in addition to the RNA primer
DNA II nuclease will then arrive and break down the DNA segments
Smaller segments which then are digested by flap endonuclease
PCNA acts as a clamp and allows DNA polymerase to move
Very specific polymerases deal with errors
DNA polymerase Kappa
DNA polymerase Beta
Remove any kind of mismatched bp because of benzokirene (that is not what it is)
Involved in base excision repair
If one mismatched pair will be removed and put in correct bp
DNA polymerase eta
Removes thymine dimers that occur from sunburns and such
Translesion polymerases
Work on original DNA strand rather than on the replicated strand
Daughter strand of other template strand used as template
Unlikely to have errors as unwinding is usually when errors occur
Will stall otherwise
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Transposable elements
Chapter 12
Gene expression
Only time that the information of what a gene possesses is available
Prokaryotes
Transcription is the first process
Two major aspects
- Proteins recognize these sequences and carry out the process
Transcription also involves unwinding of DNA just like replication
But does not copy DNA into DNA
Copies DNA into an RNA
(No change to the DNA during this process)
Unwinding still where errors are most likely (replication still has higher likelihood of error)
Definition of Gene?
Unit of heredity usually
For gene expression
a transcriptional unit
Two terms
Sense strand (coding strand)
Template strand (anti-sense strand)
Template is 3'-> 5' :
RNA are complimentary to the template strand (no uracil in DNA)
Will be similar to the coding strand
Central dogma of genetics
DNA-RNA-Polypeptide
Proteins are then tools of gene expression
- DNA sequences provide underlying information
Sequences along the DNA
Promoter sequence
Promotes transcription
Site where transcription factors come and assmeble
Regulatory factors also made which are a type of transcription factor
RNA polymerase binds after (in the case of prokaryotes)
Points out the start site of transcription
Upstream of exact nucleotide where transcription begins
Sequence on mRNA
at 5' end of prokaryotic mRNA
Ribosomal binding site
Structural genes
Actual transcription process
- Elongation
- Initiation
- Termination
Transcription factor allows RNA polymerase (holoenzyme made of many polypeptides) to bind to and identify promoter sequence
Binds at one end causing denaturation at other end
Similar to DnaA boxes
One segment of promoter sequence unwinds DNA when RNA polymerase and promoter sequence bind
Adding on RNA nucleotides based on nucleotides present in DNA
There is a termination sequence but that is not what causes the termination
Rather it is the instability between the RNA and the DNA strand that signals termination
Often times in prokaryotic genes the promoter sequence may control several gene sequences
Polycistronic DNA
Only a small percentage of uniquely repetitive sequences are structural
Transcribes but also translates
Large number of genes make RNA that do other functions
What are the most common RNA other than mRNA
Ribosomal
Transfer
Signal recognition
Secretory proteins start synthesis in cytosol then continue and complete on rough ER
Signal recognition proteins decide whether to send these proteins to the ER or finish in the cytosol
What are less common RNA other than mRNA
Small cytoplasmic RNA (SCRNA)
Help in secretion of proteins
Rnasep ribosome
Large subunit acts as ribozyme involved in transfer RNA modification
SnRNA
Part of spliceosome (small nuclear DNA)
SnoRNA
involved in modification of ribosomal RNA synthesis
Viral RNA
+1 is the first nucleotide copied into mRNA
(nucleotide upstream of +1 will be -1 (no 0))
Major group sequences
At -35 there is TTGACA
AT -10 there is TATAAT
First site read by transcription factor
Very strong binding between transcription factor, RNA polymerase, and TTGACA causes great stress on -10 region
hydrogen bonds in -10 break forming the open complex
RNA polymerase and transcription factors move to the open complex and synthesize small RNA transcript
(test run and see if there are any issues with sliding)
After test run transcription factor falls off and RNA polymerase continues
If there are any modifications in the sequence the transcription factor will go back and forth many times
Slows down transcription from usual rate if every two seconds to once every 10/15/8 minutes???
Core Enzyme= 5 subunits
Two alpha subunits of the RNA polymerase catalyze the attachment of transcription factor onto the RNA polymerase
Two Beta subunits ( beta and beta') catalyze phosphodiester bond s between nucleotides
Omega keeps the two betas and the two alphas together
Sigma factor
One subunit (S)
Influences the function of RNA polymerase
Has quaternary structure
Tertiary structure
Quaternary structure
Domains that occur with the ultimate 3d structure of an individual polypeptide
Motifs
Randomly coiled, super secondary loops, etc.
Identifies -35 subunit
Closed complex
Sliding of RNA polymerase begins at +1 nucleotide
Template is 3' to 5'
Synthesis is 5' to 3'
Copying of DNA into RNA
As copying of DNA takes place it forms a transcription bubble
length of about 17 bp (open complex)
Area behind will wind back
Area ahead will unwind via topisomerase II
Rate of synthesis about 43 bp per sec into prokaryotes
Two different kinds
Rho dependent
Rho independent
In mRNA there is a small sequence called Rho utilization substance
Rut
Rho protein comes and attaches to rut
acts similarly to helicase
Trying to catch up to open complex
A stem loop is formed where termination sequence is present along the DNA
stem loop will cause stall since any sort of structural modification stalls RNA polymerase
Rho catches up
Separates the hydrogen bonds between DNA and RNA
(no rut is present)
Stem loop will still stall
Many Uracil present at the 3' end
Unable to hold DNA RNA hybrid together
Sometimes addition of protein called nus A but not common
Only present along the DNA
Eukaryotes
Transcription
Problems with eukaryotic transcription
- Larger organisms
- cellular complexity
- multicellularity
Nuclear DNA is transcribed by three different RNA polymerases
RNA polymerase II
RNA polymerase III
RNA polymerase I
Synthesizes all of the rRNA aside from 5sRNA
All of the structural genes and RNA that are parts of spliceosomes
All of transfer RNAs and 5sRNAs
While DNA Polymerase looks like a thumb with fingers RNA polymerase looks like open jaws
DNA enters through the open mouth
Triphosphates come in through the open pore
Pyruphosphates are released and monophosphates form an ester bond
same as in the case of DNA replication
Catalytic subunit
A lot of magnesium and nucleotide pore
Clamp
Rudder
Right handed wall allows DNA to move through the clamp and control rate of synthesis
Dissociates DNA from RNA nine nucleotides after RNA is formed
Core promoter
TATA box (TATAAAA)
Much less than the 50 bp of prokaryotes
There is still a transcriptional start site up stream
Upstream from that there is usually a regulatory sequence (50-100 bp)
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Two types
Enhancer
Reulatory
Promoter site mutation cannot be overcome as it is necessary for transcription to occur
cis effect
One mutated gene for an activator can be overcome by a regulator and vice versa
Trans effect
differences
Cis effect comes from the DNA
Trans effect comes from the protein
Transcription factors
TF IID
TF IIA
First transcription factor to identify TATA box
Has certain proteins that can identify (TATA box binding proteins)
Will regulate the binding of transcription factor IID with TATA box
Can be put in either order
TF2B
will recruit RNA polymerase II w/ TF2
TFIIH and TFIIE
Once these are assembled initiation is over
TF2H has a polypeptide that does the function of helicase and ATP and Kinase activity
helps switch from initiation to elongation by hydrolyzing ATP & phosphorylation a specific sequence in the carboxyl terminal domain within the RNA polymerase (a 7 amino acid sequence tail repeated 52 times in Eukaryotes (similar to tandem repeats).
Once carboxyl terminal is phosphorylated TFIIB TFIIE and TFIIH all detach from the RNA polymerase
5th and second serines are phosphorylated
TFIID remains attached to TATA box preventing another round of transcription
When all bind it creates stress forming an open complex
Basal transcription apparatus
TFIIF
Remains attached to RNA polymerase during first part of elongation
One very long protein seen in all transcription processes
Mediator
does not bind to the DNA
Mediates the effect of regulatory factors on the rate of transcription (especially influences TFIIH and its capacity to phosphorylate carboxyl terminal domain)
Silences gene expression
(TFIIH cannot fall off)
Termination
It is still the instability between DNA and RNA that causes termination
Sequence 500-2000 bp downstream from AAUAAA will stop termination
LOOK FOR THIS SEQUENCE TO DETERMINE THAT IT IS A EUKARYOTE
Two models to explain Eukaryotic termination
Allosteric Model
Torpedo model
500-2000 bp past poly A either elongation factors drop off or termination factors hop on
PolyA signal sequence
Results in the dissociation of DNA,RNA polymerase
Similar to the Rho dependent model in prokaryotes
When RNA poly passes 500-2000 past poly A an exonuclease hops on the RNA and starts digesting nucleotides in RNA
5'-3' direction
Catches up to RNA poly II and causes dissociation
RNA modification
Not as simple as in Prokaryotes
For normal reasons but additionally several steps of regulation
Absence of colinearity between DNA and RNA sequence (there is colinearity in prokaryotes
Sequences (introns are lost in transcription from pre RNA to mRNA
Splicing exons
RNA modification that takes place in the nucleolus
Nucleolus has two functions
- ribosomal subunits are assembled
2Genes that deal with rRNA
Trimming
Transcript has sequence for three different rRNAs
RNA polymerase I catalyzes synthesis of ribosomal subunit
Endo and exoonucleases come and remove sequences between the three
The "s" is added to the names of RNA in reference to the density found in centrifusion (sedimentary coefficient)
Transfer RNA takes place in the nucleus
Also made from a large transcript
Many enzymes present
First enzyme comes at 5' end
endonuclease called Rnase P
Two parts RNA (ribozyme) one part protein fractal
Starts at middle, makes cut and starts digesting
second endonuclease
Rnase 2
removes 170 bp sequence
third endonuclease
Rnase D
Digests until it reaches consensus 5'cca3' end using ester bond (universal amino acid attached at 3' end for all tRNA)
Transfer RNA is known as an adaptor molecule
(reading nucleotides) Assembles sequence of amino acids that is then attached to adenine
Adapts to nucleotide sequence
Some tRNA do not have this sequence
Additional enzyme (transfer RNA nucleotidal transferase) brings particular sequence and then assembles (primarily attaches to amino acid)
Splicing (kind of)
mRNA will hybridize with split DNA faster than the other strand of DNA
First will form hydrogen bonds with first exon but will then be unable to form hydrogen bond with the introns
Will push the intron. The intron will then form loop.
of these loops= # of introns
Second strand of DNA will be able to form hydrogen bonds with these loops of DNA but not with the exons which are bound to mRNA
Where this 2nd strand of DNA cannot bind to DNA (over the bound exons) R loops form
These are called R loops
= # of exons
Experiment
Uses formamide to separate DNA and allow mRNA to hybridize
Intron Splicing (for real)
Group I
Group II
Can be seen in Bacteria, Archaea, Eukaryotes, Organelles
Spliceosomes
Mostly Eukaryotic organelles
Intrinsic Splicing
Do not need proteins
Self splicing (act as own ribozyme)
Remove intron RNA and link Exon RNA by a phosphodiester bond
Guanosine comes from outside and binds to intron sequence
Guanine plus sugar
Third carbon will bind at first nucleotide of the 5' end of the intron (3' end of exon)
Causes first cut
Then the 3' end of the exon will catalyze the cutting and splicing of first exon with the second exon (ester bond between exon1 and exon 2)
Two exons are connectedd
Intron is completely broken down
Nothing comes in as in the case with Guanosine in Group I (no external nucleophilic molecule)
Within the intron there is an adenine
The orientation will be such that second carbon of sugar will react at 5' end of intron and form laureate
may have evolved from intrinsic splicing
Once laureate forms and turns (twists around itself) the 3' end of the intron will break off
Exon will catalyze the formation of the ester bond between exon 1 and exon II
Exon 1 also catalyzes the removal of the intron at 3' end
Sometimes maturase catalyzes
RNA polymerase II will be involved in spliceosome complex
Two components form the spliceosome complex
rRNA
small nuclear proteins
snRNPS (small nuclear RNA and set of proteins)
Single complex contains several of these
hold pre mRNA so that intron/exon boundaries are well defined
Allows introns to be removed and exons spliced
U1 SNRP
U2 SNRNP
Gu site at 5' end of intron binds here
U1 will induce attachment of
recruits three other SNRNPS
U5
U4
U6
dimer
Cause intron to be pushed outside
Causes stress and results in first break at the 5' end of the intron
5' end will then attach to adenine at branch site
laureate will form bringing intron sequences closer together causing U1 and U4 to fall off
Metallic ribozyme as it has two magnesium atoms inside
will catalyze the removal of introns and attachment of two exons together
SNRNP complexes and introns are then pushed out and degraded
Alternative splicing
Introns may increase stability and may allow for alternative splicing
Lower eularyotes see very little alternative splicing
Higher eukaryotes such as ourselves see 70% of genes have alternative splicing
More cellular complexity
Some exons must be present no matter what but some are organ dependent
DNA sequence then makes pre mrna
Those present in all mRNA
Constitutive mRNA
For example
Although there are different types of tropomyosin they all have the constitutive genes for contraction
Factors that regulate spliceosomes
Splicing factors
One example is the SR proteins
two terminals
n terminal
c terminal
positive
negative
high concentrations of serine (S) and arginine (R)
Capable of identifying protein part of snrnp
protein uses to bind to RNA
3' and 5' refer to the carbon of the nucleotide which is being added to
DNA can only be added on the 3' carbon
DNA replication often results in two intertwined molecules called catenases
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Creates holoenzyme
as opposed to deoxyribose
Histone formation occurs during the S phase
Form into octamers
Mixture of old and new upon replication
How are the ends of Eukaryotic chromosomes replicated
Telomerase
3' overhang
at telomeres
12-16 nucleotides in length
5' 3' template
loops are meant to be 100-200 bp
too short to form RNA/DNA primer
DNA polymerase
Only synthesizes in 5'->3' direction
Cannot link together first two individual nucleotides
If this problem was left unsolved telomeres would get progressively shorter (full length of DNA would not be replicated)
loops are 100-200 bp long
5'->3' template
blood cell and skin cell telomeres would get small
rapid proliferation
telomerase RNA component (TERC)
Acts as template to help synthesize the overhang
enzyme
Telomerase reverse transcriptase
Comes and binds first
Three nucleotide segment of RNA comes and binds to complementary DNA
Synthesizes a 6 nucleotide long DNA repeat strand
moves six to the right and attaches to last three then synthesizes another 6
Translocation
Continues until enough to make a loop
Still a 3' overhang but dealt with
uncontrolled cell division diseases
a lot of telomerase activity in these cells
Focused on as cure
Length does decrease in normal cells
some segments of DNA can move around the genome
Endosperm
Triploid
two haploid nuclei from the mother one from the father
McClintock experiment
Corn
Colorless Kernel
Colored Kernel
Dominant
Plant with one copy of the dominant allele
Found kernels that were white/ colorless and some colored here and there
hypothetically should be all colored
Colored gene on chromosome 9
Found interesting sequence in between
dissociation sequence
highly transposable element
can go to different parts of genome within the corn
sequence split aside by presence of dissociation sequence
caused kernel to be colorless whenever present
dissociation sequence not has gone past that sequence when colored
two ways in which sequences can be inserted
Simply as a DNA sequence
transposon
RNA intermediate
DNA copied into an RNA
RNA reverse transcribed to DNA
Then inserted
Retro transposon
Many more copies can be made
RNA sequences are shorter
chances much lower than retro transposon
transposon gene
two sequences on either side (inverted sequences)
identical sequences opposite and below on either side
Next to these (outside) direct sequences
these are identical
simple transposons
highly repetitive if small moderately repetitive if larger
Retrotransposons
long terminal repeats (LTR)
Share much in common with retro viruses
integrase
have viral origin
Have reverse transcriptase gene
non LTR retrotransposons
inverted and direct repeats are not usually present
Can start from any eukaryotic gene
something like a reverse transcriptase but usually functions more like an endonuclease
genes capable of identifying sequences within DNA and making cuts
Alu family of genes has no viral origin for example
If no integrase or reverse transcriptase or endonuclease
non-autonomous
Look at dissociation sequence
Cannot move anywhere
without activator sequence (AS)
transposase will bind to the inverted sequence
proteins bind to identical sites on DNA and loop the DNA
cuts and then can be inserted elsewhere
simple transposition
have other genes not neccesary
these are also on retro transposons
allow to be identified
cuts at staggered points
pushed and then inserted and gaps are resynthesized
resynthesized are direct repeats
You have two copies made during unwinding
we have endopolyploidy cells
One can insert itself ahead of the replication fork and be replicated again (on strand where there was no translocation)
most important enzyme
Integrase
Reverse transposase
May encode gene that functions both as reverse transcriptase and endonuclease
Whenever mRNA transcript is made
two modifications
5' methylguanosine cap
stabilizes mRNA
3' end (200 nucleotides with adenine as nitrogenous base)
PolyA tail
Allows RNA to find out specific locations in DNA that have similar sequence
Look for sequences complementary
binds to Ts in DNA sequence
seen in RNA DNA hybrid primer
creates tilt
selfish DNA
Occurs due to duplication not translocation
Can happen during non-allelic homologous crossing over
Chapter 13
Initiation factors (green= prok purple=euk)
Recognize the 7 methylguanosine cap at the 5' end of mRNA and facilitates the binding of the mRNA to the small ribosomal subunit
EIf4
Prevent the association between the small and large ribosomal subunits and favor their dissociation
IF1, IF3
eIF1,eIF3,eIF6
Promote the binding of the initiator tRNA to the small ribosomal subunit
IF2
eIF2
Helps to dissociate the initiation factors, which allows the two ribosomal subunits to assemble
eIF5
Elongation Factors
Involved in the binding of tRNAs to the A site
EF-Tu
eEF1
Required for translocation
EF-G
eEF2
Recognize a stop codon and trigger the cleavage of the polypeptide from the tRNA
RF1, RF2
eRF1
GTPases that are also involved in termination
RF3
eRF3
No termination to be concerned about with eukaryotic replication
Capping
occurs when translation is only 20-25 bases long
If there is no capping then the mRNA will not be released from the nucleus
- Capping first takes place on the first nucleotide of triphosphate (RNA 5' triphosphotase is first enzyme made (going to remove one of the phosphates).
- Then GTP will arrive and bind to phosphates (brought by guanylyl transferase (then two phosphates are released as pyrophosphate and are left with a Guanine monophosphate attached)
- Then attach methyl group to the seventh position of the guanine (nitrogenous base (via a methyl transferase)
7 methyl guanosine cap
seen by gatekeepers to allow passing
Exonuclease and endonuclease look for this if non existent will be digested
Adenine nucleotides at 3' end
Rna Editing
Involves deletion or replacement of a specific type of nuceotide
Apoliprotein B
In liver
In small intestine
B 100
B 48
Change from CAA to UAA results in shorter protein
Different types of amino acids
long chain
aromatic
benzene ring
aliphatic
Alkaptonuria
tyrosin metabolism
Causes ochronosis
considering homeogentisic acid oxidase
reccessive
Remember this is similar to phenyl kaptonuria
gene is defective of making this enzyme
neurospora (mold stuff)
What causes these to be mutated
methionine is needed
synthesized by biochemical pathway
a bunch of enzymes
plate divided into five sections
wild type grows fine
Each progressive mutant strain had mutated enzyme coding genes
Progressive strains unable to code for next progressive enzyme in the pathway
When the enzymes were added independently of coding they were able to code for the methionine at the end of the pathway and the neurospora would be able to grow
conclusion- genes code for specific polypeptide
not just enzyme
not necessarily a protein either
proteins must be functional
translation
language of genetic codes converted to language of amino acids
How many nucleotides make a code?
4 different nucleotides
3 make a code
4 to the power of three
64 codes are possible
61 code for amino acids
3 are responsible for stop codons
UAG
UGA
UAA
Results in methionine
start codon does code for a polypeptide
Always methionine
AUG
There only 20 amino acids
More than one code must then code for an amino acid
Eukaryotic versus prokaryotic
Codes are degenerative
third nucelotide known as wobble base
does not follow chargaffs rule in transfer RNA
sometimes 2nd or first but rarely
for example GGU GGC GGA GGG
All code for glycine
In eukaryotes and prokaryotes
What is the structure of an amino acid
alpha carbon
r group
Positive terminal (n) and negative terminal (c) never switch
and hydrogen
switch places to get specific amino acid
such as polar group having a hydroxyl group
charged has an acid amine group
polypeptide chains have directionality
Long chain of amino acids called poly peptide
Carboxyl
amino
many peptide bonds
connects carboxyl with amino via release of water
types of structure
Primary
arrangement of amino acids based on codes in mRNA
proteins never found in primary structure
not funcitonal
Secondary structure
Have two types of modifications
alpha helical
Beta pleated
hydrogen bonds between adjacent amino acids with bend formed
Hydrogen bonds between amino acids far apart in long string
this is what linus pauling used to find structure of DNA
Only hydrogen bonds
Tertiary structure
strength determined by this
Hydrogen
Ionic
hydrophobic exclusion
All non polar amino acids are pushed inside associated in internal structure
Van der Walls
covalent
only disulfide
Quaternary structure
proteins formed of multimeric units
Proteins from more than one polypeptide
all of the same bonds in tertiary
Dissociation
They can move without being harmful
Denaturation (all proteins can unfold in harmful environment)
Chaperones help renature
heat shock proteins
Different functions of proteins
Workhorses of cell
immune
transport
storage
Enzymes
this is what we highlight
Catabolic
Anabolic
help in synthesis (use of energy)
first enzyme of glycolysis
Transfer RNA
Adapts to codes present in mRNA
Assembles amino acids based on those codes
direct interaction with proteins
brings amino acids based on codes read on the mRNA
Adapter
Amino acid attaches to the CCA sequence at 3' end
always attached to adenine
34th base will correspond with wobble base and not follow chargaff rule
isoacceptor stem
76 nucleotides in tRNA
there will be three stem loops
middle stem loop will have anti codon
hydrogen bonds with codes on messenger RNA
ester bond
20 of these
Some bases are modified in tRNA
adenine deaminated to give inosine
Sometimes methylated
uradins can be modified to give pseudo uradin
pseudo uradin labeled p
These modification allow for enzymes that attach amino acid to be attached to tRNA
Amino acyl transfer rna synthetase
Also can help bind to anti codon
150 nucleotide modification
click to edit
20 of these
requires second reading (Reading of tRNA)
2 steps
Activation of amino acid
ATP
Amino acid
aminoacyl transfer RNA synthetase
Both attach to enzyme
ATP is hydrolyzed
Pyrophosphate released
AMP will attach and charge amino acid
tRNA will attach
AMP is released
now you have charged transfer RNA
then enzyme and transfer RNA (plus amino acid = charged) wiill be released from one another
amino acid reads tRNA
bound to second or more commonly third carbon of adenine
RIbosome
two subunits
rRNA
Protein
assembled separately in nucleolus
bulk of ribosome
plant cell
mitochondrial
chloroplasts
cytosol
Prokaryotes
Euaryotes
70s coefficient
small subunit
large subunit
80s
30s
50s
16srRNA
23srRNA
34 proteins
21 proteins
5 srRNA
large sub unit
small subunit
60s
40s
18srRNA
33 proteins
5.8 srRNA
28 srRNA
49 proteins
large sub unit sits on top of small sub unit
large sub unit
A site
P site
E site
Aminoacyl site
Peptidyl site
Exit
1st TransferRNA that does process of translation enters here
Methionine is always the amino acid bound to the transfer RNA so that is why it is marked
All following tRNA enter through A site
mRNA binds to small subunit of ribosome first
modified methionine binds in proks
mimic will cause end
comp to sd
also cap binding
comes with GTP
23 3 S catalyzed movement
ribozyme
weird ribosomes
16s recognizes shine delgano
23s catalyzes peptide bonding
Rf1
Rf2
UAA, UAG
class one
UAA, UGA
Associated with primase and helicase (primosome)
forming the replisome
decatenation