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70 Cards in this Set
- Front
- Back
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What are cis-acting elements?
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promoter sites that RNA polymerase recognizes to start transcribing.
Called cis because the sequences are on the same molecule of DNA as the gene being transcribed. |
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What are trans-acting factors?
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aka transcription factors
proteins that activate or repress transcription Trans because are not on the DNA molecule ( a totally different species) |
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what does RNA polymerase do?
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binds to promoter sites
melts and unwinds DNA recognizes termination sites interacts with transcription factors (proteins) makes mRNA, tRNA and rRNA |
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RNA polymerase utilizes a metal ion in the active sites, cierto o falso?
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cierto
3 Asp participate one metal is bound to RNA polymerase the other metal is bound to NTP and leaves with PPi |
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What are the subunits of the E. Coli RNA polymerase?
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a2BB'sigma
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What's the difference between the holoenzyme and the core enzyme of RNA Polymerase?
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holoenzyme has the sigma subunit still attached.
core enzyme is the other subunits once the sigma subunit has left after it has done it's job locating the promoter site. contains the catalytic site |
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What's the difference between the holoenzyme and the core enzyme of RNA Polymerase?
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holoenzyme has the sigma subunit still attached.
core enzyme is the other subunits once the sigma subunit has left after it has done it's job locating the promoter site. contains the catalytic site |
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What is the sigma subunit's job?
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locate promoter sites and initiate transcription.
recognizes certain nucleotide bases in promoter sites on DNA. -10 and -35 regions are where the sigma interacts with template DNA. |
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What is the sigma subunit's job?
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locate promoter sites and initiate transcription.
recognizes certain nucleotide bases in promoter sites on DNA. -10 and -35 regions are where the sigma interacts with template DNA. |
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What do the metal ions in the catalytic site of the RNA polymerase do?
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The ion positions the 3'OH in the best way to make it's Nu attack on the a Pi of the incoming NTP.
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What is footprinting?
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DNA is marked with 32P
There is a control group and a group with binding proteins. Then they add DNAseI to make a bunch of different cuts. The sample with the binding protein compared to the control cuts will tell you where the promoter sites are that the proteins bound to and protected that strand from the DNAseI. |
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Which direction does RNA polymerase build the nacent RNA in?
What's the -35 promoter sequence in pro's? |
5'--->3'
TTGACA |
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What is the -10 promoter sequence in prokaryotes?
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TATAAT
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Why would it be logical that the promoter site / pribnow box would be a collection of A's, and T's?
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because these bonds are lower in energy and easier to melt the DNA because they only form 2 H bonds.
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What's the coding DNA strand?
What's the template DNA strand? Which one does the nacent RNA match? |
Coding DNA is base-paired to the template DNA. (sense + strand)
Once the dsDNA is unwound the RNA builds base pairs with the DNA template strand (anti-sense - strand) RNA matches coding DNA except that it has U instead of T. |
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What does anti-sense strand mean?
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The template strand is moving in the 3'-->5' direction so it is called anti-sense. Referred to in (-)
The coding DNA strand is referred to as the sense strand is and has + designations. Because RNA matches DNA coding strand it's first nucleotide transcribed is referred to as +1 |
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What is the UP element?
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upstream element adds strength to the signal of the promoter
located -40 to -60 range binds a-subunit of RNA Poly |
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What are the 3 different kinds of sigma subunits?
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sigma 70 is the regular one
sigma 32 is the heat shock genes sigma 54 is the nitrogen-starvation promoter |
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With regard to RNA Poly unwinding capabilities:
how many bp's does it unwind? how many turns of B-DNA is this? |
17 bp of DNA are unwound
This is 1.6 turns of B-DNA |
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What role does topoisomerase II play in the unwinding action of RNA poly?
What is the unique aspect to its own negative feedback regulation? |
makes negative supercoils to allow for the unwinding that takes place during transcription.
Topo II increases the efficiency of transcription at distally located promoter sites, except where the gene for itself is going to be transcribed. THis way we don't have too many topo II's running around and coiling everything up. |
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What's at the starting end of nascent RNA in pro's since it doesn't need a primer?
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pppA or pppG
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what happens to the strength of RNA Poly core enzyme binding to DNA once sigma takes off?
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Binds strongly to DNA. aids in processity, accuracy and recognizing termination sites.
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How many bp's long is the newly formed RNA - DNA hybrid helix?
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8 bp and about 1 turn of double helix
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How many bp's are "melted" in transcription bubble?
How fast does the bubble move? |
17 bp's
170 A / sec = 50 nucleotides / sec The hybrid continually turns to keep the 3' OH nu in the proper position. |
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cierto o falso? RNA poly's show some proofreading nuclease activity?
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cierto.
They make more mistakes 1 in every 10-4 or 10-5 but it's not transmitted to progency so its cool. |
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What is the stop signal for termination of transcription?
What's the other termination signal? |
palindromic GC region followed by AT region
These guys make the hairpin turn which is followed by a poly-U chain. p (rho) proteins are additional factors that cause termination. |
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Why poly U to halt transcription?
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The U's have a weak bond with the DNA so it will leave off the DNA and exit.
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What does hexameric p protein bind to specifically?
How does it work? |
ssRNA
C (poor in G) sequencees bind p ATP hydrolysis makes it like a helicase that catches up to the bubble and breaks the hybrid helix. |
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How would a mutation in the the expression of p protein impact protein synthesis?
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p cannot terminate RNA so the transcriptions would be longer and thus mess up gene expression and synthesis of proteins.
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How does post-transcriptional modifications work in Pro's?
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there isn't a great deal of it. Often mRNA is translated right after it gets transcribed because remember all of this takes place in one compartment.
tRNA and rRNA are made after some cleavages to nascent RNA (E. coli has 3 rRNA's and a tRNA that get excised from primary RNA) |
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What are the 3 rRNA's and 1 tRNA that gets made from primary RNA in pro's?
What are the enzymes that cleave the primary RNA to make these guys? |
16S, 23S and 5S rRNA
just tRNA, no number ribonuclease III (5, 16, 23 S rRNA's) ribonuclease P (tRNA) |
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What is added to the end of tRNA's in pro post-transcriptional modification?
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CCA tail
This polymerase does not use a DNA template to add the tail. |
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What type of RNA base and ribose modifications happen post-transcriptionally in pros?
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rRNA can be methylated
tRNA made from Uridylate into pseudouridylate or ribothymidylate |
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In Euk's where does transcription and translation take place?
What impact does this have? |
transcription--nucleus
translation--cytoplasm multi-cellular organisms use differential transcriptional reg to create different cell typees and enables intricate regulation of gene expression |
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What are the three ways how RNA is handled in Euk vs. Pro to lead to greater complexity?
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Nuclear membrane
differential and complex transcriptional regulation extensive RNA processing |
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Where does the pre-mRNA get processed?
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pre-mRNA is processed and spliced In the nucleus
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What are the 3 RNA Euk polymerases?
Where are they located? What do they do? |
RNA Poly I
nucleoli transcription 18S, 5.8S, 28S rRNA genes RNA Poly II nucleoplasm transcription of mRNA and small RNA genes RNA Poly III nucleoplasm transcription of 5S rRNA and tRNA genes |
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Where and what does RNA Poly I do?
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nucleoli transcription
18S, 5.8S, 28S rRNA insensitive to a-amanitin toxin |
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Where and what does RNA Poly II do?
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nucleoplasm transcription
mRNA and small RNA genes This is the guy with the carboxy tail domain (CTD) filled with SER that gets phos'd for the transcription to begin toxin a-amanitin inhibits |
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What and where does RNA poly III do?
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nucleoplasm transcription of 5S rRNA and tRNA genes
Inhibited by high concentrations of a-amanitin toxin |
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What are the promoters involved with RNA Poly
I in Euk? |
rInr (ribosomal initiator element has TATA-like sequence)
UPE (upstream promoter element--lies 150-200 bp upstream) |
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What are the promoters involved with RNA Poly II?
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many combinations
enhancers Inr TATA box DPE (downstream promoter element) |
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What are the promoters involved with RNA Poly III?
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conserved sequences that lie within the transcribed gene
Type I in 5S rRNA has A Block and C Block Type II in tRNA have A Block and B block |
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What is the protein associated with RNA Poly II that phos's the CTD?
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TFIIH
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Describe the TATA box promoter for RNA Poly II.
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most common. Those genes that get expressed rapidly or more frequently tend to be TATA
It is located between -30 and -100 |
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Where is the Inr located for RNA Poly II?
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Between -3 and +5
Defines the start site and increases transcriptional activity |
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Where is the DPE located on RNA poly II?
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between +28 and +32
Usually present with Inr when there is no TATA box |
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What are some additional regulatory sequences besides Inr, TATA or DPE that function with RNA Poly II?
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CAAT box and GC box
-40 to -150 These guys can be located on template DNA strand in EUK ( the -35 region in PRO's are on the coding strand) |
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What is the differences between the promoter sites in Pro's vs. Euk's?
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The promoter sites in Pro's directly attract RNA Polymerase, whereas the promoters in Euk's attract other proteins (TF's) who then call in the RNA Poly.
Also, there is way more variety in promoter order, location, combination in Euk. |
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What is the main role of TF's?
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To make a solid spot for RNA Poly II to come along, sit down in the right spot and start translating.
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Describe TFII?
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Has the TBP (tata box binding protein) which asymetrically and tightly binds to TATA box.
It is saddle shaped and thus bends and unwinds DNA when it binds through PHE hydrophobic interactions |
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Describe what happens after TBP on TFIID binds to the TATA box
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TFII:
A B F RNA Poly II E H=phosphorylation of CTD= go elongation. most factors release as RNA Poly II goes on to do its thang. |
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Talk about why heat shock transcription factos show gene differentiation.
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There are lots of different elements upstream of promoter sites that get recognized by a lot of different proteins that will congregate to call polymerases to that specific spot based on environmental or developmental needs for specific gene expression.
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Describe how Ig enhancer allow RNA Polymerase access to specific genes.
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a particular enhancer is only effective in certain cells
Ig enhancer only works in B cells Burkitt Lymphoma and B cell Leukemia has a translocation which grabs proto-oncogene myc and attributes the Ig enhancer qualities to it so the myc gene gets enhanced |
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Where does the 40S small ribosomal subunit for a ribosome come from?
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the 28S rRNA made by RNA Poly I
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Where does the 60S large ribosomal unit on a ribosome come from?
Where does the other large part of the ribosome come from? |
28S and 5.8S rRNA from RNA PolyI
5S rRNA from RNA Poly III |
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What is it that extensively modifies pre-rRNA destined to make the ribosome?
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snoRNP's
many small nucleolar ribonucleoproteins --takes place in nucleolus --bases and riboses get methylated --SSU's process pre-rRNA and help put together the ribosome --mature rRNA and cleavage + the ribosomal proteins lead to the mature ribosome |
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What is the enzyme that cleaves the 5' end to get tRNA rolling?
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RNAse P
Then they get the CCA cap and have splicing and base and ribose modifications These guys are highly processed. |
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Describe how a cap gets put on a tRNA.
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a 5' ppp (A or G) releases Pi
The remainding PPi attacks the a-Pi of GTP to form a 5'-5' PPPi link |
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What's methylated in cap 0?
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The N-7
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what's methylated in Caps 1 and 2?
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adjacent riboses
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What gets caps?
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mRNA
not tRNA nor rRNA |
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What cleaves off the AAUAAA sequence?
What adds the Poly A chain of ~250 A's? What's the role of the poly A tail? |
an endonuclease
poly A polymerase tail increases m RNA stability and translation efficiency |
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What happens if mRNA doesn't have a pioly A tail?
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It still gets transcripted but it is not nearly as effective as a template for protein syn
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Do you keep introns or exons?
What does an intron begin and end with? |
keep the exons and splice together.
introns start with a GU and end with AG |
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What are the 5' and 3' splice consenses in euk genes for splicing together extrons?
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5' agGUaagu
3" 10 pyrimidines (U/C) and ended with ncAG |
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Describe a spliceosome.
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60S complex of snRNP's, splicing factors and mRNA precursors
A- 2' OH in branch site attacks the 5' end of the first part of intron to create a transesterificaiton reaction |
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Describe the lariat intermediate.
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After the A-2'OH attacks the intron it releases the first exon whose 3' end goes to grab the 2nd exon's 5' end.
So you have the transesterification bond between the branch A and the attack of one exon on the other |
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How much energy is used up in a splice?
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None, it is accomplished through two transesterification reactions rather than utilizing GTP or ATP
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What are all the U's involved in splicing?
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snRNA's
U1 binds the 5' splice site U2 binds the branch site to get it to attack where U1 is U5 Binds to U1 and U2 (Picture the intron looping out here) U4 masks U6 U6 cuts! |
