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106 Cards in this Set
- Front
- Back
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What proportion of the eukaryotic chromosome is DNA? Protein?
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~50% DNA
~50% Protein |
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What are histones?
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small (11-22kDa)
very basic (20-25% lys + arg) proteins |
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What are the core histones?
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the four histone proteisn that form a disk that the DNA wraps around
-2 copies each of H2A, H2B, H3, H4 |
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What is H1?
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H1 is a "linker" protein between the "core" histones
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Are histone proteins highly conserved?
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Yes, H4 from cows differ by 2 AA from H4 of peas.
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How much DNA warps around each histone core?
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dsDNA wraps around the histone core ~1.65 time, or 147bp of DNA.
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What is a Nucleosome?
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The histone core with 1.65 rotations of DNA wrapped around it, and 20-60bp of linker DNA.
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What are NTD?
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NTD, or N-terminal domains, are the long, AA strands that extend out of the nucleosome from the histone core.
NTDs make up 25-30% of the total mass of the histone core. NTDs are UNstructured in the crystal structure |
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How/where do histone proteins and dsDNA interact?
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The lys and arg in the histone core interact with the negatively charged phosphate backbone of the DNA in the MINOR groove.
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How to histone proteins interact? (How is the histone core formed?)
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The H3 and H4 histones form heterodimers, then join together to form a tetramer with 2 mol each of H3 and H4.
The, H2A and H2B form heterodimers in solution. H3, H4 tetramers then bind with DNA. Then 2 H2A, H2B dimers join the H3, H4, DNA complex to form the final nucleosome. |
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How do NTD modifications affect nucleosomes and DNA compaction?
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Acetylation and phosphorylation in general will decrease the NTD's affinity to the negatively charged backbone of DNA, which makes an acetylated or phosphorylated NTD nucleosome more accessible for transcription machinery.
Methylation of the NTDs, however, causes condensation and binding of specific proteins, making it harder to transcribe the DNA in the nucleosome. |
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What are bromodomains? chromodomains?
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Bromodomains are protein domains which can be incorporated in various enzymes that bind to specific acetylated sites on NTDs.
Chromodomains are protein domains which can be incorporated in various enzymes that bind to specific methylated sites on NTDs. These two domains could be incorporated into enzymes to allow, say, transcription machinery to bind to a type of site. The fact that certain enzymes only have certain domains allows the cell to regulate when transcription occurs by methylating or acylating NTDs in nucleosomes at various times in the cell. |
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Are eukaryotic chromosomes supercoiled?
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In short, yes.
Although eukaryotic chromosomes are not ccc, covalently closed circles, they are sufficiently long such that they are topologically constrained. Plus, it is unclear if the ends of chromosomes are attached to the nuclear matrix or not. Wrapping DNA around histones is equivalent to negative supercoiling, and Eukaryotes have Type I and II topoisomerases. |
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How is DNA replicated?
Conservative? Semiconservative? |
Semiconservative, one parent strand is used to make one daughter strand, and the other parent strand is used to make another daughter strand.
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Is chromosomal replication uni-directional?
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NO! Chromosomal replication is bi-directional. Two sets of replication machinery move in opposite directions of one another.
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In which direction does replication occur?
5'-3'? 3'-5'? |
5'-3'! Why? Because of the nature of phosphodiester bonds. These bonds are most easily added in the 5'-3' direction, so why would DNA ever add them in the 3'-5' direction?
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Qhat is the subsrtate for DNA replication?
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dNTPs, or deoxyribo Nucleotide TriPhosphates, where N = A, C, G, or T.
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Please explain how a dNTP is added to the 3' end of a DNA strand.
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the 3'-hydroxyl group of the growing chain carriest out a nucleophilic attack on the α-phosphate of the incoming dNTP, forming a new phosphodiester bond.
the β-phosphate and γ-phosphate remain attached to each other, and are freed to float away from the replication fork in the form of a PyroPhosphate, PP, molecule. |
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What enzyme is required to allow DNA polymerase to add dNTPs to a naked DNA template strand?
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Primase!
Primase adds 5-10 NTPs, or riboNucleotideTriPhosphates, to start a DNA primer strand, which is complimentary to the DNA template strand. |
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What is an Okazaki fragment?
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An Okazaki fragment is the short primer and DNA molecules, base paired to the template strand, on the lagging strand of DNA.
This fragment is usually about 2kbp long. |
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Is DNA replication continuous?
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No! DNA replication is semi-continuous. the leading strand of DNA is synthesized almost entirely as one long strand of DNA with a 5'-end primer. The lagging strand of new DNA, however, is synthesized in small fragments called Okazaki fragments.
Also, these two processes occur at an overall equal speed, since both DNA polymerase III's, one for the lagging and one for the leading strand, are attached to the same Replisome. |
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What functional domains does primase have?
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RNA strand initiation domain
NTP addition domain |
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What functional domains does DNA Pol III have?
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dNTP addition domain
exonuclease domain |
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What is helicase and how does it work?
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Helicase is the enzyme used in replication which looks like a donut with a center hole only big enough for one strand of DNA to fit through. This forces DNA to split as the helicase moves on one strand in the 5'-3' direction, the complimentary DNA strand is split apart from its other strand in the 3'-5' direction.
This unwinding action is powered by hydrolysis of ATP, and creates positively supercoiled DNA in front of it. This means, without unwinding, evenatually the DNA would be too compact to allow helicase the ability to unwind more DNA. So, topoisomerase is required at the other end of the dsDNA to unwind it as it is wound by helicase. NOTE: Since DNA synthesis occurs in the 5'-3' direction, the lagging strand of the DNA must be on the strand of DNA which helicase is attached too. |
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What is SSB?
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SSB is single stranded binding protein.
SSB binds to ssDNA to prevent reformation of dsDNA after the helicase unwinds the it. SSB is very cooperative. In other words, the liklihood of adding SSB to ssDNA is much higher if the SSB is added next to another SSB. |
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What does DNA Ligase do and what is its function in DNA replication?
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DNA ligase "ligates" DNA molecules together. In replication, the nicks left behind by DNA Pol I are sealed by DNA ligase.
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What is the function of RNAse H in DNA replication?
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RNAse H removes the RNA from RNA-DNA hybrids (hybrid = H in RNAse H). However, it can only cleave bonds between ribonucleotides, therefore one ribonucleotide will be left attached to the newly synthesized strand. It might also be prudent to node that at this time there is a gap of open space where the RNA primer used to be.
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What are the functional domains of DNA Pol I?
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Polymerase Activity
3' to 5' Exonuclease - Fix Errors 5' to 3' Exonuclease - Remove RNA or DNA in "front" or primer |
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What does DNA Pol I do in replication?
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DNA Pol I removes the final primer ribonucleotide left behind by RNAse H, but can not join the final 3' end of one piece of DNA to the 5' end of another piece of DNA. This leaves a nick in the DNA.
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Please list the proteins required for replication and their general roles.
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Helicase (DnaB)
-Melts parental DNA, interacts with PolIII and Primase Primase (DnaG) -Synthesizes RNA primers DNA PolIII -Synthesizes DNA starting at RNA primers SSB -Binds ssDNA template cooperatively, prevents reannealing and hairpins RNAse H -removes RNA primers DNA Pol I -Removes DNA primers and fills gap with DNA DNA ligase -Seals nicks between okazaki fragments |
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What proteins make up the Replisome?
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2x DNA Pol III
3x sliding clamp, (the ϐ-subunit) DNA helicase Primase 2x tau-proteins γ-complex |
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What are the essential domain DNA pol III domains for DNA replication?
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the DNA polymerase
the 3' to 5' exonuclease |
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What is the holoenzyme?
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The Holoenzyme is the major enzymes that are required for replication. The holoenzyme is made up of the following parts:
2 Core polymerases (DNA pol III) 1 Clamp 2 Clamp Loader (gamme plus tau subunits) |
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Where does replication action occur in the DNA pol III structure?
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DNA pol III looks like a hand. The incoming dNTP is bonded to the primer strand at the palm in between the fingers and thumb of the hand shaped DNA pol III.
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How does the polymerization of the incoming dNTP occur?
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the proper dNTP, base paired with the template strand, interacts with two divalent metal cations.
one metal ion binds to and helps position the dNTP and neutralizes its charge. The other metal ion activates the 3'-OH grp of the primer strand by essentially turning it into 3'-O(-), which makes it very reactive. The O- of the primer strand then attacks the alpha-phosphate of the dNTP, creating a phosphodiester bond breaking the alpha to beta anhydride bond, leaving the pyrophosphate unattached to the new 3' end of the primer strand. |
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How does DNA pol III recognize and add only correct dNTPs in DNA replication?
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The conformation of DNA pol III's DNA polymerase active site is such that only correctly hydrogen-bonded dNTPs will have their alpha-phosphate group close enough to the primer strands 3'-OH grp to undergo the creation of the phosphodiester bond.
Also, there are amino acids positioned around the active site such that riboNTPs 2'-OH grp will not fit into the active site such that the alpha-phosphate can bind to the activated 3'-O- of the primer strand. Also, the O-helix in one of the fingers of DNA pol III has Tyr, Lys, and Arg side groups which are positioned to do the following. -the Lys and Arg staalize the neg charged beta and gamma phosphate groups of the incoming dNTP -the Tyr stabalizes the dNTP by armoatic stacking interactions if and only if it is properly hydrogen bonded to the template strand's unpaired base. |
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What do some DNA pol III's do that can increase accuracy of DNA replication by 100x?
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proof reading!
Some DNA pol III's can identify if the last paired base is mismatched, which causes the pol to activate exonuclease activity at the base of the fingers before resuming DNA synthesis. |
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What is the beta subunit of the holo enzyme?
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The beta subunit of the holoenzyme is the Clamp.
The Clamp has a 35 A donut hole which completely surrounds DNA or DNA-RNA hybrid, and freely slides up and down the DNA. The Clamp is tethered, however, to the PolIII core which acts to hold the PolIII core to the DNA. |
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Whatis the gamma subunit of the holoenzyme?
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The Clamp Loader!
There is only one clamp loader per replisome, which is an integral part of the polymerase complex. It recognizes the 3' end of the primer/DNA hybrid, and uses several ATP to open the Clamp and Load it onto the primer/DNA hybrid (double stranded region). This means, in theory, the clamp loader only acts once on the leading strand, but acts about every 2kbp on the lagging strand. |
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What is DnaB?
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DnaB is Helicase, which is a hexamer (6 identical subunits). Helicase requires DnaC to load it.
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How is helicase's action controlled?
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Helicase is stimulated by its interaction with the tau-subunits of the holoenzyme, so that when it gets ahead of the holoenzyme, it slows down to where the holoenzyme moves faster than the helicase. This allows the two to basically stay together during replication.
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What is DnaG?
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DnaG is the gene that codes for Primase. Primase is recruited and stimulated by helicase to lay down a 5-10 ribonucleotide primer. Then, the primase dissociates from the helicase. Thus, this is not a processive process, and must be done independently every 1-2kbp on the lagging strand.
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What events occur at the replication fork?
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Helicase can contact both primase and tau-subunits.
Helicase recruits primase to open the origin of replication Primase lays down 5-10 ribonucleotide primer Clamp Loader recognizes primer Clamp loader loads the clamp Helicase recruits primase continually to initiate lagging strand synthesis (though not processively) |
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How is topoisomerase incorporated in DNA replication?
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Either Topoisomerase I or II can relax the positive supercoils in the DNA introduced by helicase. Without topoisomerases, DNA replication would quickly stop.
Gyrase is believed to be the most important enzyme for relaxing DNA in front of the replication fork |
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What are the chemical interactions used by DNA ligase?
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Step 1: ATP is hydrolized and AMP is bound by an anhydride bond to the 5' alpha phosphate group in the DNA nick (or single strand break). Then, AMP is hydrolized and a phosphodiester bond is formed between the 5' alpha phosphate and the 3'-OH group.
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What is OriC?
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OriC is the replication initiation site in E. coli's genome. There is only one replication site in prokayotic genomes.
This region contains 9 bp repeats, which are binding sites for DnaA, and 13 bp repeats, which become single stranded when DnaA-ATP binds. |
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How does replication initiation occur in E. coli?
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Step 1: Multiple DnaA-ATP proteins bind to the repeated 9bp sequences with the OriC.
Step 2: The binding of these DnaA-ATP to the 9bp repeats causes the 13bp repeats to open into a region of ssDNA. Step 3: A DnaB and DnaC (hilcase and helicase loader) associate with the DnaA-bound origin. Step 4: DNA helicase loaders catalyze the opening of the DNA helicase protein ring and placement of the ring around the ssDNA at the orgin. Loading of the hlicase leads to dissociation of the loader form the helicase, which activates the DNA helicases. Step 5: The helicases recuit primases, which lay own their first primers. Step 6: The newly synthesized primers are recognized by the clamp loader components of two DNA pol III holoenzymes. The sliding clamps are assembled onto each RNA primer and leading strand synthesis is initiated by one of the two core DNA pol III enzymes of each holoenzyme. Step 7: After each holoenzyme has moved about 1000kbp, another primer is synthesized on each lagging strand template and a sliding clamp is loaded. the resulting primer:template junction is recongnized by the second DNA pol III core enzyme, resulting in lagging trand synthesis. Step 8: Leading strand and lagging strand synthesis are not initiated at each replication fork and continue to the end of the template or until another replication fork from an adjacent origin of replication is reached. |
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How is new DNA different from old DNA?
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An enzyme known as Dam, or DNA Adenine methyltransferase, recognizes the sequence
5'GATC 3' 3'CTAG 5' and methylates the adenines. The methyl group sticks out into the major groove of the DNA so it can be recognized by other enzymes. In newly synthesized DNA, the template strand has already been methylated and the new strand has not. This can also be recognized by enzymes. |
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How do cells control replication?
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In one of two ways:
1)The cell controls concentration of DnaA-ATP in two ways. -the concentration of DnaA-ATP is proportional to the mass of the cell, so only a cell large enough to be ready to replicate DNA can undergo repliation. -DnaA-ATP is hydrolized into DnaA-ADP causing it to dissociate fro the OriC. DnaA-ADP is does not work as an initiator and is converted back into DnaA-ATP very slowly. 2) Control of access to oriC -DnaA-ATP binds only to fully methylated DNA -The protein SeqA binds to hemimethylated oriC preventing DnaA from binding and Dam methylation. |
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How does termination occur in prokaryotes?
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Ter sites bound with Tus, anti-helicase, proteins can only be passed by replisomes in one direction.
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What is a catenane?
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A catenane is two cccDNA looped around each other like to rings. Topoisomerase IV is believed to be the primary enzyme involved with detaching the two newly replicated cccDNA from each other.
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What is different in eukaryotic and prokaryotic replication?
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In eukaryotic replication
-the replisomes move more slowly, probably due to the complex chromatic structures -there are many more origins of replications -eukaryotes must unpack and repack chromatin throughout replication. -the end replication problem. |
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What is the end replication problem?
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The last few bases in the newly synthesized strand of DNA is not filled in because DNA pol I must have a 3'-OH group to bind incoming dNTPs to. Since one doesn't exist at the 5' end of the newly synthesized DNA, there is no way to finish this strand with cimplimentary base pairs.
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What are telomeres?
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The ends of eukaryotic chromosomes are called telomeres
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How is the end replication problem fixed?
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Telomerases, a ribonucleoprotein DNA polymerase (telomerase is a reverse transcriptase), adds ssDNA to the 3' end of the template strand using its own built in RNA template. Then, the normal replication machinery can add a primer to the new DNA and close the gap between the new primer on nonsense telomeric DNA and the genomic DNA at the original 5' end of the primer strand.
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What are the differences between RNA and DNA?
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RNA contains riose instead of 2'-deoxyribose
RNA contains uracil instead of thymine RNA is usually ss instead of ds, however the bases of ssRNA often base pair in between regions of the single strand of ssRNA. |
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What is alkaline hydrolysis?
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Alkaline Hydrolysis is a chemical reaction that RNA undergoes in which one end of the phosphodiester bond between two ribonucleotides is proken, and the phosphate bonds with the 2'-OH group, which doesn't exist in deoxyribonucleic acid. Then, the phosphate group can unbind, from either the 3' or 2' Oxygen, leaving a broken RNA with a 5'-OH grp, and one with a 3'-Phosphate group.
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What kinds of "special" interactions stabalize hairpin loops?
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Base to phosphate hydrogen bonding
G-U base pairing (as well as other known basepairing) Stacking interactions |
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What are some important structures of a tRNA?
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the acceptor arm
the anticodon loop |
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How can RNA be denatured and what in RNA is denatured?
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The weak non-covlent interactions of RNA are denatured by:
-High Temperature -Hydrogen bonding reagents - Urea -Increase in hydrophobicity - Methanol -Decreased salt concentration |
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What are the 3 types of RNA, what are their functions, and what types of RNA polymerases transcribe them?
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Messanger RNA, mRNA, is the template for protein synthesis. mRNA is transcribed by RNA pol II in eukaryotes and RNA pol in prokaryotes
Transfer RNA, tRNA, is the adapter between mRNA and amino acids. tRNA is transcribed by RNA pol III in eukaryotes and RNA pol in prokaryotes ribosomal RNA, rRNA, is the heart of the ribosomes that synthesize protein. rRNA is transcribed by RNA pol I in eukaryotes and RNA pol in prokaryotes |
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What types of RNA are found in the cell other than the 3 major types?
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Major types: rRNA, tRNA, mRNA
Other: -Regulatory RNAs which anneal to mRNAs. -Catalytic RNAs, i.e.ribozymes, usually affect other RNAs. |
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What are the steps of transcription?
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1. RNA polymerase binds to the promotor to form a closed complex
2. "Melting" produces an open complex with a single stranded bubble. 3. Transcription begins but only short RNAs are made. 4. Eventually the polymerase "escapes" from the promotor and enters the elongation phase. 5. When the full length RNA has been transcribed the RNA is released and the polymerase dissociates (termination) |
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What are the subunits of E. coli's RNA polymerase holoenzyme?
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2x alpha
beta beta prime omega sigma |
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What are the sizes of the subunits of E. coli's RNA polymerase holoenzyme?
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alpha 37kDa
beta 150 kDa beta prime 155 kDa omega 10 kDa sigma_70 70 kDa |
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What is known in MCB 250 about the structure of RNA polymerase?
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RNA pol looks like a pincer claw. beta prime is the top part and beta is the bottom. The DNA is split inside the pincer, and the template strand follows along the top of the channel and the non-template strand moves along the bottom.
The top template strand runs by the active site, which polarizes the 3'-OH bond to make the 3'-O- which is reactive. The sigma protein runs along the length of the pincer, interacting with the core RNA polymerase and the DNA at various locations. |
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How is transcription initiated in prokaryotes?
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The sigma factor in RNA polymerase recognizes the promoter region of a gene, the -35 and -10 promotor regions using the sigma_4 and sigma_2 domains respectively, and the alpha C-terminal domain interacts with the UP-element near the promoter sequences if the UP-element is available. The RNA pol at this point is a closed complex.
Then, the RNA pol unwinds the DNa around the promotor to form the RNA pol open complex. This clsoed to open complex requries a major change in conformation. Then, approximately 10 small ribonucleotides are produced, this is called abortive transcription, until finally the RNA pol "escapes" from the promotor. Note: Neither the -35 or -10 promotor sequences, nor UP element are required for transcription of a gene, however they increase the likelihood of gene transcription if they are closer to the ideal conserved promoter sequences and location. |
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What are the promoter regions for sigma_70 for transcription in prokaryotes and where are they located?
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The -10 site is TATAAT
The -35 site is TTGACA The -10 and -35 site have 17 to 19 bps in between them. |
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How does the alpha subunit of RNA polymerase interact with DNA?
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The alpha CTD interacts with the UP element, and the alpha NTD binds to the RNA polymerase. The UP element strength can increase the likelihood of transcription. This allows for activators with strong UP elements to bind to DNA to activate gene transcription.
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What do repressors do?
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Repressor proteins bind to operators in the promoter to physically stop RNA pol from binding to the DNA
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How does elongation in transcription occur in prokaryotes?
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As the RNA pol exits the initiation and enters the elongation phase, it undergoes another conformation change. Then, the sigma factor eventually dissociates (no particular timing matters).
As RNA pol synthesizes RNA, elongation factors load onto the polymerase, which can do thinks like increase processivity, accuracy, and/or aid in termination (i.e. NusA) Note: the rate of transcription varies on the frequency of pausing. |
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What are the two methods of transcription termination which occur in prokaryotes?
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There are two methods are:
-rho-independent or intrinsic termination -rho-dependent termination |
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What is rho-dependent termination and when does it occur?
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Rho-dependent termination occurs when the rho-protein, bonded to the newly synthesizing RNA, runs into the RNA polymerase. This forces the RNa pol to hop off the DNA at the next pause site. In particular, this pause site is known as the Rho termination site, and there is a sequence of RNA that aids in biding of the rho protein, which is known as a rut site.
The rho can only bind to a region of RNA that does not have ribosomes on it. Rho's APTase activity allows it to move along ssRNA ina 5' to 3' direction Rho's RNA-DNA helicase activity allows it to seperate RNA-DNA duplexes. Rho-dependent termination can be stopped by Antitermination |
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What is intrinsic termination and how does it occur?
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Intrinsic termination occurs when a strong inverted repeat section of DNA is separated by weak bases (A, U), and the inverted repeats are followed by a strand of Uracils bases.
This essentially forms a terminator hairpin, which signals to RNA pol to pause. The poly-U section of recently synthesizes RNA, which is the only RNA inside the RNA pol at this time, is weak enough that it is very likely to separate from the RNA before the RNA has a chance to resume transcription. |
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What is antitermination?
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Antitermination occurs when various sites in RNA are recognized by auxilliary proteins that create a complex which is NOT subject to Rho-mediated Termination and which ignores all but the strongest intrinsic terminators. This involves a NusA protein which binds to the RNA pol and other auxiliary proteins, and tells RNA pol to ignore Rho. The sequences of RNA that are important here are known as Box A and Box B.
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What is polarity?
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Polarity describes a gene that has a nonsense mutation which stops transcription of a polycistronic gene.
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How are genes orientated in cccDNA?
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They can be on either strand of dsDNA, and can overlap each other, and go in different directions. However, they tend to all move away from the ori.
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What does "S" mean in say the 16S ribosomal subunit?
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The S stands for Svedberg Units, and is a sedimentation coefficient. Svedberg designed a method for seperation of molecules based on how quickly they move towards the bottom of the tube when centrifuged. This speed is known as the sedimentation velocity.
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How many genes in E. coli code for rRNA and how many code for tRNA?
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7 rRNA genes code for 16s, 23s, and 5s rRNAs.
79 genes code for 46 tRNAs. |
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How are rRNA and tRNA processed after transcription?
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Commonly, the genes that transcribe these RNAs are polycistronic, which results in one long rRNA. But, we dont want to translate these genes, we want specific regions of the rRNA cut out to be used in larger ribozymes. This is done using specific RNAses, which cut at various sites on these rRNA and tRNA transcripts.
Then tRNA's 5' end is snipped short by RNaseP. The 3' end is also processed by particular RNases. |
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What is RNaseP and what does it do?
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RNaseP is a ribozyme which snips the 5' end of tRNA short. RNaseP's catalytic domain is RNA alone.
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How is Eukaryotic transcription different from prokaryotic transcription?
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Eukaryotes have 3 RNA polymerases with specialized functions.
Eukaryotes mRNAs are monocistronic (no operons) Transcription and translation in eukaryotes are spatially and temporally seperate in eukaryotes. mRNA is 5' capped and polyadenylated, so both the 5' and 3' ends are modified. splicing of pre-mRNA is freqently required to obtain a mature mRNA Only after all modifications and splicing are complete is the mature mRNA released from the nucleus to the cytoplasm. |
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how is the structure of the eukaryotic RNA pol's and prokaryotic RNA pol similar?
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They both have the same general structure, only Eukaryotics has many more proteins.
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What is important about RNA pol II's CTD?
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RNA pol II's C-Terminal Domain is made up of a large number, 25-50, sequences of 7 amino acid repeats (heptad repeats).
The sequence is -Tyr-Ser-Pro-Thr-Ser-Pro-Ser- The Ser residues can be phosphorylated and the phosphorylation site of the CTD provides signals that direct binding of various proteins. |
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What do the promotors of Eukaryotic genes look like (for transscription)?
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They contain many many binding sites for dozens of proteins, which interact with each other.
There are two general types of sequences, those that start with TF for transcription factor, and TBP sites for TATA binding protein. |
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How does transcription in eukaryotes occur generally?
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TFIID binds to DNA, with the TBP that is bound to it recognizing the TAT box. Then, other proteins join up which signals the RNA pol II to hop on. Then, some proteins bond to RNA pol, whihc creates a DNA bubble and changes the conformation of RNA pol. Then, the CTD of RNA pol II gets phosphorylated and RNA pol II lets go of the pre-initiation complex to begin elongation.
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What do the general transcription factors do for initiation of transcription in eukaryotes?
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They function together as the sigma protein seen in prokaryotes, as well as they aid in opening the DNA bubble necessary for transcription.
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Why is modeling of transcription in eukaryotes different in vivo than in vitro?
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In vivo, the DNA is wrapped up in nucleosomes, so it take many more proteins to unwrap, transcribe, then rewrap the DNA.
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What do the mediator complex and HAT proteins do in vivo?
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The mediator complex is required to initiate transcription in vivo. It mediates between proteins bound to DNA and the pre-initiation complex.
The HAT (histone acetyl transferase) protein is involved in taking apart nucleosomes in vivo. |
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What kind of mRNA processing occurs in Eukaryotes?
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5' cap
3' polyadenylation splicing |
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What is the 5' cap?
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The mRNA is capped as soon as the 5' end of the RNA is made. The cap is a 7-methyl guanosine bonded to the 5' end of the mRNA with a 5' to 5' phosphate bridge.
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What does the 5' cap do for mRNA?
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-important for splicing
-protects the mRNA from RNases -Enhances the efficiency of translation, the cap binding protein -Required for efficient transport from the nucleus. |
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How does polyadenylation occur?
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The CstF (Cleavage Stimulating Factor) and CPSF (Cleave and Poly A Specificity Factor), which are both bound as a complex to the CTD of RNA pol II transfer to the mRNA when the poly A signal seqence in the RNA is seen. They then snip the RNa and CstF dissociates.
The CPSF stays and PAB, or Poly A Binding Protein, binds to the end of the mRNA. As are then added to the end of the protein and poly A binding proteins bind to the tail. |
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What is the purpose of the poly-A tail?
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It protects the mRNA from RNases, which increases its stability
-when the PolyA tail is finally lost, the mRNA is degraded. It also enhances translation -the PAB somehow does this |
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Only Pol II transcripts (mRNAs) are Capped and Spliced in Eukaryotes. Why?
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The capping and splicing machinery interact with the carboxy terminal "tail" of RNA Pol II which is absent in RNA Pol I and RNA Pol III
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How does the splicing reaction occur?
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The phosphate from the 3' end of the exon, or where ever the RNA is spliced, breaking the bond between the 3'-OH of the 3' end of the exon and the 3'-O-Phosphate of the 5' end of the intron, and binding the 3'-O-Phosphate to the 2'-OH group of an adenine at a specific location in the intron. Then, the 3'-OH of the 3' end of the exon is bonded to the 3'-O-Phosphate of the 5' end of the next desired exon, which cleaves the intron from between the two exons.
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What catylizes the splicing reaction?
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Although this can occur in the cell naturally, sometimes a splicosome is built on splice sites to aid in splicing.
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What is the splicosome made of and how do these components interact with the RNA?
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The splicosome is made up of 6 snRNAs (small nuclear RNAs); Ua, U2, U3, U4, U5, and U6, and a bunch of proteins.
The snRAs combine with proteins to make snRNPs (small nuclear ribonuclear proteins). These snRNPs interact with RNA to splice introns out. The U2 grabs the 2'-OH of the specific adenine, then U4 leaves the spliceosome, and the 3' end of an exon's 3'-O-Phosphate bond is cleaved to form a 5'-O-Phosphate to 2'-OH bond. Then, the 3' end of the exon's 3'-OH is bonded to the 5' end of another exon's 5'-O-Phosphate. |
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How is the timing of various protein additions to mRNA from RNA pol II controlled by the eukaryotic cell?
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The phosphorylation of specific AAs of the heptide repeat causes binding or dessociation of various proteins to the CTD of RNA pol II.
No phosphorylation means the RNA pol II is in the preinitiation stage. if the 5-Serine is phosphorylated, then the RNA pol II is in the promoter escape phase, and the capping enzyme is recruited. When only the 2-Serine is phosphorylated, the RNA pol II is in the elongation phase, and the splicing factors are recruited. |
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When is the mRNA transported out of the nucleus?
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When it is covered in a specific set of bound proteins and has a 5' cap and polyA tail.
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Why is alternative splicing important?
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Alternative splicing explains why the human genome contains about 20k genes and 100k+ proteins.
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Is ATP required to form or use splicosome?
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Lots of ATP is required to form the splicosome, but no ATP is required for the splicosome to act.
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What components are required for translation in prokaryotes?
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mRNA
tRNA Aminoacyl tRNA synthetases Ribosomes Initiation, Elongation, and Termination factors |
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What is the RBS?
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The RBS, or Ribosome Binding Site, is a seqence like GGAGG which complimentary base pairs with the 16s rRNA. The sequence is located about 2-9 bases from the 5' end of the mRNA.
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What is the acceptor stem of tRNA?
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it is a ..CCA-3' end, which extends out further than the 5' end of the tRNA. This sequence is conserved in all tRNA.
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Are all bases on tRNA C, G, A, and U?
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No, there are quite a few modified bases found in tRNA.
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