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223 Cards in this Set

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Terminators
trigger the elongating polymerase to dissociate from DNA and release the newly made RNA chain
Rho-Dependent is a what?
Terminator
Rho dependent requires what to terminate? and what does it do?
Rho protein. it binds a single stranded RNA as it exits the polymerase and uses ATPase to pull it away from the DNA template and polymerase
What two rules does Rho follow?
It first bind Rut sites (that are not secondary structues) Then if a transcript is being translated (bound by ribosome) it will aviod that transcript
The only initation factor bacteria needs?
Sigma factor
Initiation factors that Eukaryotes use?
General transcription factors (GTFs)
core promotor
Minimal set of sequence elements required for accurate transcription by the RNA Pol II
The TATA box is what? and is bound by? and how?
Where it all binds. TFIID. (Transcriptional factor) Through its TATA binding protein (TBP)
What does the TBP do?
distorts the TATA box and recruits TFIIE and TFIIH and the mediator Complex
What does TFIIH do?`
provide the necessary helicase activity and uses ATP
In orfder for the RNA Pol II to elgongate what must it do?
Get ride of the TF. (once it starts elongating it will be leaving proteins behind)
How does this happen?
By phosphorylation of the C-Terminal domain (CTD) activated by a kinase found in TFIIH
Which TF also has a kinase?
TFIIH
When transcribing Chromatin, activators do what?
help recruit polymerase to the promotor, stabilizing it's binding site
What does the mediator complex do during chromatin transcribing?
Mediates the activators recruitment, and also bind the CTD of the RNA Pol II
Yeast and human mediator complexes contain ? subunits. ? of which are homologous
>20. 7
Steps to elongation of Transcription
Pol II sheds the initiation Factors and they are replaced with elongation factors.

CTD recruits factors for processing. (BRINGS IT ALL TOGETHER AT THE SAME TIME)
What processing factors are recruited by the CTD
RNA capping enzyme on the end of the RNA

RNA Splicing machinery assembles next

Then the cleavage and polydenylation factors.
What stimulates the bonding of these factors?
The phosphorylation of Serine
How does the RNA pol get past histones?
Spt16/SSRPI work together to disociate the H2A-H2B dimer.

The RNA Pol then can roll through

Then Spt16/SSRPI then reassemble the nucleosome with the help of SPT6
What is the first modification of new RNA?
It is capped at it's 5'
How does this happen
Removal of a phospahte at the 5' end

guanine is added

Methyl group is added to the 7N position
Final modification of new RNA?
Polyadenylation at its 3' end
What is crucial for this polyadenylation at its 3' end?
CTD
Steps in the Polyadenylation : first step
The enzymes cleavage and polyadenlyation specificy factor (CPSF) and the cleavage stimulation factor (CSF) move from the CTD of RNA Pol to the poly-A Sequence (at the begining of the new RNA)
Second step
Other proteins are recruitted and RNA is cleaved
Third step
CPSF binds the Poly-A signal and Poly-A Polymerase (PAP) is recruitted to add more adenines to the 3' end of the mRNA
Fourth step
around 200 adenines are added, poly-A binding proteons begin to bind the long stretch of poly-A
Fifth step
All poly-A recruitment factors and enzymes dissociate from the mRNA
LECTURE 16
LECTURE 16
exons
coding
introns
non coding
Difference between pro and eukarytotes when it comes to gene structure?
exons are interupted by introns in Euk
transcription of eukaryotic genes produce?
pre mRNA
After the pre mRNA is produced, the ? are removed by ?
Introns. RNA splicing
exons longer or introns longer?
introns
How does it relates to complexity?
More introns, more complex. more introns make the organism more diverse bc there is more control in splicing
intron-exon boundary
site that will be spliced
Branch point. what is it, found where.
point where the spliced 5' introns makes the lariet. entirely in the intron
First step in splicing
the 2'-OH A site attacks the Phosphate at the G site.
what was that result?
the freed 5' end of the intron is joined to the branch site
Second step
the 3'-OH group of the recently freed exon attacks the phospahte at the 3' splice site
The result
the two exons are spliced together and it releases an intron lariet
trans-splicing
splicing between two separate exons
what happens?
same mechanisms, only a Y shaped structure is produced
What mediates the trans-esterfication rxns? and whats its major functional unit?
Splicesome. RNA
RNA has how many and what are they called
five. U1 U2 U4 U5 U6. snRNAs
Each of these is paired with several what? new name?
Proteins. snRNPs
what does U1 do
labels the 5' splice site
U2FA?
binds the 3' splice site
exons
coding
introns
non coding
Difference between pro and eukarytotes when it comes to gene structure?
exons are interupted by introns in Euk
transcription of eukaryotic genes produce?
pre mRNA
After the pre mRNA is produced, the ? are removed by ?
Introns. RNA splicing
exons longer or introns longer?
introns
How does it relates to complexity?
More introns, more complex. more introns make the organism more diverse bc there is more control in splicing
intron-exon boundary
site that will be spliced
Branch point. what is it, found where.
point where the spliced 5' introns makes the lariet. entirely in the intron
First step in splicing
the 2'-OH A site attacks the Phosphate at the G site.
what was that result?
the freed 5' end of the intron is joined to the branch site
Second step
the 3'-OH group of the recently freed exon attacks the phospahte at the 3' splice site
The result
the two exons are spliced together and it releases an intron lariet
trans-splicing
splicing between two separate exons
what happens?
same mechanisms, only a Y shaped structure is produced
What mediates the trans-esterfication rxns? and whats its major functional unit?
Splicesome. RNA
RNA has how many and what are they called
five. U1 U2 U4 U5 U6. snRNAs
Each of these is paired with several what? new name?
Proteins. snRNPs
what does U1 do
labels the 5' splice site
U2FA?
binds the 3' splice site and helps the branch point binding protein (BBP) bind to the branch site
What replaces the BBP and binds to the branch site and extrudes (unpairs) the A site?
U2
What three are then added? and what do they do?
U4 U5 U6. pulls together and produce lariet
which two are held closer together?
U4 and U6
which one leaves now and what replaces it? and why?
U1 leaves because it's only job was to define the boundary and U6 has pulled the link together and replaced it
who does the annealing?
U6
what happens when U4 leaves?
U2 and U6 can now bind
what happens when U2 and U6 bind?
the form the active site and break the 5' end and attaching it to the branch site
which helps to bring the two exons together?
U5
Most common spilcing?
pre mRNA splicesome (with the spliceosome
2nd most common?
Group II self spilcing. same mechanisms, doesn't need a spliceosome.
Least common splicing?
Group 1 self-splicing. uses secondary structure to fold the RNA
What is it called when the exon sites are skipped?
exon skipping
pseudo splice sites
when other close splice sites are mistakenly read as splice site and it is cut in half
loading on splicing machinery during transcription would be one way to improve...
the accuracy of splice-site selection
What do SR proteins do?
bind to exonic splicing enhancers (ESEs) within the exons
What does this do?
The SR proteins bound to the ESEs interact with the splicing machinery and recruit it to nearby splice sites.
SR proteins are responsible for recruiting which of the U1 U2 U4 U5 U6 proteins?
U1 to the 5' and U2AF to the 3'
LECTURE !7
LECTURE 17
degeneracy
amino acids are encoded by more than one codon
synonyms
codons that encode the same amino acid
mutations in the first nucleotide positions will often,,,
will give the same amino acid
pyrimadines in the second position code for ...
hydrophobic amino acids
purines in second position encode for
polar amino acids
Transition mutations in the third position ...
rarely changes an amino acid (50%)
highly purified tRNAs can
bind several different codons
Which anticodon can bind the most codon bases?
Inosine (3)
which end of the anti codon is not as restricted?
5'
this allows it to?
form hydrogen bonds with several different bases at the 3' end of the codon
the codon is in the ? direction
3'-5' direction
codons are read by ribosomes in the ? direction
5'-3'
Codons are ? and mRNA contains ? gaps
non-overlapping. zero.
mRNA is translated in a ? from the ?
fixed reading frame. start (AUG). meaning since they don't overlap, the nucleotide can be translated in any of the 3 reading frames
Missense mutaion
changes a codon specific for one amino acid to a codon specific for another amino acid`
effect length of poly peptide?
no
nonsense (stop) mutation
changes a codon from one amino acid to a stop codon
effect polypeptide length?
yes. makes it shorter than it would have been.
Frameshift mutation
where one or a small number of base pairs are inserted or deleated and alter the frame
effect polypeptide lenght?
maybe. you could get a stop codon earlier down the road
Two ways to reverse mutations?
reverse back mutations / suppressor mutations
Reverse back mutaions
where an altered nucleotide is changed back to its original arrangement
suppressor mutations
muatations that occur at different locations on the chromosome that supress the change
Two kinds of suppressor mutations
Intragenic and intergenic
Intragenic suppression pg. 534
mutations occuring in the original gene, but at a different site in the gene
Intergenic suppression
mutation occurring in another gene?
Where the coordinated action of over 100 proteins and RNAs yeild a single protien
Transcription
Translation machinery has four compnents
mRna
tRNA
Aminoacyl tRNA synthetases
Ribosome
mRNAs
provide the template codons that specify the order of amino acids
tRNAs
the physical inteface between the mRNA and the added amino acids
Aminoacyl tRNA synthetases
couple the amino acids to the proper tRNA
ribosome
Composed of protein and RNA. (snRNSPs) coordinates recognition of the mRNA by tRNAs and forms the peptide bond between individual amino acids and the growoing polypeptide chain
Open reading frames
non overlapping stings of codons that secify different protein sequences
What is a real ORF>
Has the longest strain, with both a start and stop codon
polycistronic mRNAs? Pro or euk?
Have multiple ORFs and encode for more than one protein.

PRO
In PROs , in order for translation to occur what has to happen first?
A ribosome must be recruited to the mRNA
How is it recruited?
Through the Prokaryotes Ribosome binding site (RBS)
Where is the RBS located?
upstream of the start codon
Difference in Eukarytoes?
the mRNA can be modified to help direct translation without the RBS
How?
capping on the 5' end recruits the Ribosome
Start codon is also called?
5'-AUG-3'
Where is the Kozak sequence? what does it do? Pro or Euk?
Before the start. increases translation efficiency. Euk
Major difference between Pro and Euk transcription and translation
Transcription and translation happen in the same place in Prokarytoes and not in Euk
Where does transcription happen in Euks?
Nucleus
Where does translation happen in Euks?
Cytoplasm
Is translation faster in Pro or Euks? why?
Pros, because it all hapens in the same place
How does transcript/lation happening in the same place help make Pros go faster?
Because the ribosome can procede with translating the mRNA as it emerges from the RNA Pol and multiple ribosomes can translate off the same mRNA
DNA Pol does the...
Ribosomes do the...
Transcription
Translation
The two subunits the compromie the Prokaryotic ribosome
50s (large) subunit
30s (small) subunit
which contains the peptide transferase center, and what does it do?
50s. it is responsible for forming the peptide bonds
Which contains the decoding center, and what does it do?
The 30s. where tRNAs read the codon units of the mRNA
What is the in tact Prokaryotic ribosome called?
70s Subunit
What are the two subunits of a Eukarytoic ribosome?
40s (small)
60s (big)
The intact Eukaryotic ribosome is called the
80s
What form of RNA is present in Ribosomes?
rRNA
HOw is the mass of each ribosome split?
Between RNA and proteins
More RNA or Proteins? why?
More proteins because they are smaller.
First step of Ribosome association
mRNA binds to the 30 subunit first followed by the initiator tRNA
second step
the 50s subunit is recruited
third
protein synthesis is started at the 5' end of the mRNA
fourht
new tRNA are added and released to grow the poly peptide
fifth
the ribosome encounters a stop codon
sixth
the polypeptide chain is released
7th
the ribosome disocciates back into a large and small
how many ribosome can translate the same mRNA?
many!
Why is this important?
cells do not have abundant mRNA (1-5% of a cells RNA)
What are the two most abundant forms of RNA?
rRNA and tRNA
New amino acids are attched to the gorwin poly peptide chain where?
At the C-Terminus (Carbon)
What is the single chemical reaction that ribosomes do?
form a peptide bond between each new amino acid and the growing peptide chain
the two charged species of tRNA that help addition of new amino acids
Aminoacytl-tRNA
Peptidyl-tRNA
First step in the addition of new amino acids
the peptidyl is present at the C-Terminus of the polypeptide
second
the amino (N) group of the aminoacyl-tRNA is brought close to the C-Terminus of the peptide-tRNA
third
the amino (N) group of the aminoacyl-tRNA attacks the C-terminus of the peptide-tRNA
the result?
and new peptide bond is formed
proteins must grow from the ? to the ? termini
N to the C termini
Peptide transferase reaction
the name of this whole process of the peptide bond being transfered from pep-tRNA to Amine-tRNA
How many tRNA binding sites does the Ribosome have? and there names
3. E, P, A
The P site
is the binding site for peptidyl-tRNA
the A site
the binding site for aminacyl-tRNA
the E site
the binding site for the tRNA that is released
What is the order that tRNA binds these sites?
P, A, E
LECTURE 19
LECTURE 19
the tRNA that binds the P site first is called what?
fMet-tRNA
why is it called fMet-tRNA
because it contains a formyl group (HCO)
Most of translation occurs with or without a Full ribosome?
Without, mainly just with the 30S
There are three ? that help translation initiation
initiation factors
how are these initaion factors labeles?
IF1
IF2
IF3
what does IF3 do?
occupies the E site of the 30s and prevents the 50s from binding
what does the IF2 do?
IF2 interacts with the IF1 and the initiator tRNA to help bind the fMet-tRNA
How does IF2 help bind the fMet-tRNA?
It uses GTPase
What does IF1 do?
Blocks tRNAs from binding the A site
Step 1 of prokaryotic Translation Initiation
IF3 bind the E site and blocks the 50s from binding
second
IF1 and IF2 bind and block the A site from binding tRNA
third
the initiator tRNA is recruited
fourth
it binds the Psite with the help of IF2
fifth
the mRNA start codon bind the initiator tRNA causing a shape change
sixth
IF3 is released and the 50s binds
7th
the creation of the 70s initiation complex causes the IF2 to hydrolysize its GTP causing IF2 and IF1 to leave
8th
Now with IF1 and IF2 gone, the A site can now bind new tRNA
first Steps to Ribosome assembly in EUKARYOTES
eIFA binds to the A site so stop the tRNA from binding to the 40s A site
second
eIF1, eIF3, eIF5 all bind the E site to prevent the 60s from binding
third
the eIF2/initiator complex is brought to the P site. the meth on this initiator tRNA is not special)
fourht
the 40s subunit is now recruited by the 5' capped mRNA
First steos to mRNA assembly in EUKARYOTES
5' cap is put on the 5' end of the mRNA
second
eIF4E recognizes the 5' capped end
third
additional initiation factors join in (eIF4G binds eIF4E and the mRNA)
(eIF4A binds eIF4G and then the mRNA)
fourht
eIF4B binds and activates eIF4A helicase activity
fifth
the activated helicase causes eIF4A to unwind any secondary structes at the 5' end so that is can bind to the ribosome
What is the name of the completed 40s ribosome combined with the mRNA?
48S pre initiation complex
REVIEW OF THE eIF's jobs
REVIEW OF THE eIF's jobs
eIFA
binds the A site- prevents tRNA from binding
eIF1, eIF3, eIF5
all bind the E site and prevent 60s from binding
eIF2/initiator complex
binds to the P site
eIF4E
recognizes and bind the 5' caped end
eIF4G
binds eIF4E (at end) and eIF4A
eIF4A
binds eIF4G and the mRNA. Also uses its helicase do to the unwinding on the mRNA
eIF4B
activates the RNA helicase activity of eIF4A and it unwinds any secondary structures
steps AFTER THE 48S preinitiation complex is formed
the complex hydrolosizes ATP and scan the mRNA in a 5'-3' direction, looking for a start codon
second
the anticodon tRNA bind the mRNA start codon,
third
all the initiation factors are released and eIF5B comes in a hydrolysizes its GTP
result?
the 60s can now bind
what does the poly-A tail do?
keep the initiation factors closely associated with the 3' end of the mRNA and helps recycle ribosomes
what does the eIF4G do?
interacts with the poly-A binding proteins and circularize the mRNA
what does EF-Tu do
escorts aminoacyl-tRNA
steps to do this
EF-Tu binds the 3' end of the aminoacyl-tRNA and prevents it from binding until it is released
EF-Tu can only bind aminacyl-tRNA when?
When it is bound to GTP
second
EF-Tu-GTP takes the aminacyl-tRNA to the A site and binds it to the mRNA codon
third
EF-Tu interacts with the factor-binding center, causing it to hydrolsize its GTP and release from the aminacyl-tRNA
1 stop to preventing incorrect aminoacyl-tRNAs to the codon
two adjacent amines help from 3 bonds and these bonds are only the strongest when there is a correct pairing, if there is not one, they dissociate.
2 way
in order for the EF-Tu to release the aminayl-tRNA into the A sopt, the anticodon and the codon just match up, if they don't it will not release it
3 way
if an incorrect aminyl-tRNA is drop in the A site, when the amine roates to leave it's peptide, if it is incorrect it will just pop off. ACCOMODATION
EF-G: steps to moving the tRNA from the A to the P
EF-G muust first bind GTP to bind to the ribosome
second
During the hybrid state, the EF-G-GTP can bind the A site (factor binding site)
third
the A site stimulates the GTP and the EF-G extends down to the bottom of the A site and pushes it to the P site, causing the P site to over to the E
fourth
Now EF-G-GDP loses its affinity and is release, along with the old P site tRNA