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79 Cards in this Set
- Front
- Back
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What is meant by saying DNA replication is semi conservative?
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This means that the daughter strand of DNA will have both one new strand and one old strand
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What are the three stages of replication?
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Initiation,
-Specific site (Origin) selected, replication forks established Polymerazation -Movement of replication forks through chromosome Leading strand – continuous Lagging strand – discontinuous Termination -Ends of DNA reached and “sealed” Separation of daughter chromosomes |
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Where is the origin of replication in E.Coli and what will bind to the origin of replication?
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The origin of replication is OrgC and the DNAa will bind to the OrgC. About twenty DNAa will bind to OrgC.
--Binding site for DNAa is a 9 bp sequence, so oriC is at least 180 bp long |
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The origin of replication must be _______
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Methylated
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How are nucleotides methylated by the DAM?
i.e where exactly does DAM methylate. |
DAM will add CH3 to the A in the GATC sequence
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What enzyme recognizes the GATC sequence in E. Coli?
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DAM-DNA adenine methylase
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What is refered to as hemi-methylated?
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Double stranded hybrid between methylated and non-methylated strands is called “hemi-methylated” .
daughter strand(s) are made using dATP (no methyl group) |
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The DNAa will not bind to hemi-methylated or to unmethylated strands so what must happen
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DNA Adenine methylase will add methyls to the strand.
The DNAa will then recruit other proteins for DNA replication such as helicase and DNA polymerase. |
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What can high levels of tension do DNA?
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This can break the phosphate backbone
Negative supercoiling – underwound Positive supercoiling – overwound |
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Helicase will melt and unwind the DNA strands, creating a _____ supercoil at the back and a _______ supercoil at the front?
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Negative
Positive |
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What are the topiosomerases that don't require ATP and which do ATP?
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DNA Gyrase (Type II)-Creates negative supercoils and this will use ATP
Type I, III, IV-Don't use ATP-relieve supercoiling through creating a single – stranded break (a “nick” in the dsDNA) |
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What inhibits DNA gyrase and this will produce what in dsDNA?
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Fluoroquinolones (ciprofloxacin) are antibiotics which inhibit DNA gyrase. Inhibition of DNA gyrase results in ds breaks of chromosomal DNA
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What DNA polyemerase is involved in DNA repair and removing RNA primers and puts down 600 base pairs a minute?
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DNA Pol I will eventually remove the RNA portion of the fragment and replace it with DNA
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What DNA polymerase is used in DNA repair and is found usually in the stationary phase?
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DNA poly II
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What DNA Poly is the fastests and will add up 1000nts/ minute before dissociating. Is a Large multi subunit complex
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DNA Poly III
“pol III holoenzyme” |
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Besides DNA Poly II what else is expressed in the stationary phase?
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DNA Poly IV
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What DNA Poly is Expressed in response to radiation damage as part of “SOS response.” Highly error prone, able to polymerize even when template strand is heavily damaged or modified.
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DNA Poly V
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How are the base pairs matched?...i.e what determines spatial position of the bases.
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The spatial location of the hydrogen donors and acceptors
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The 3’ OH on the growing chain is used to attack the _____ phosphate of the incoming dNTP
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Alpha
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What is released as replication occurs?
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Pyrophosphate (P2O7) is released
-This is a highly exothermic (energetically favored) reaction |
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Why is primase needed
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The 3’OH is strictly required for DNA polymerase. This means DNA polymerase cant bring in the first dNTP during synthesis as there is no previous nucleotide to provide the OH. There is an enzyme called primase that synthesizes a small complementary RNA to the template strand. The 3’OH at the end of the this RNA is then used to catalyze subsequent addition of a dNTP.
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The binding of the new dNTP to the 3'OH is provided by what?
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Energy for reaction provided by
triphosphate |
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Why is there a lagging strand in DNA replication?
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The strands are opening up at the same time in ONE direction. The lagging strand will not have an available 3'OH group to attack the alpha phosphate group of the incomming dNTP. So a Primase will add in a strand of RNA so it can be used for DNA replication.
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What DNA poly fill the gaps made by the ozaki fragments in the lagging strand?
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DNA Poly III
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Describe the process of filling in a lagging strand in DNA?
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The lagging strand is made creating Ozarki fragments
The DNA poly III will come in and fill in the gaps made by the lagging strand DNA poly I will then enter and use its RNAse activity and replace RNA with DNA. The DNA poly I will leave gaps between one strand of DNA to another DNA strand. This gap will be filled with ligase. DNA ligase requires ATP. |
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Single Stranded DNA is more susceptible to degradation from Nucleases, what prevents this during DNA replication?
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SSB: Single strand binding protein
Protects ssDNA from damage. ssDNA is more susceptible to nucleases |
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DNA poly III is a an alpha 2, beta 2 _____.
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tetramer
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Tau protein first binds itself to
form a homodimer. This homodimer is used to hold what together? |
DNA Poly III
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The DNA poly III works on both the lagging and leading strand, THE TAU PROTEIN HOLDS TOGETHER THE DNA POLY III, and the beta subunit forms a sliding clamp holding the DNA poly III to the DNA. What provides for the 3'-5' exonuclease activity?
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The epsilon subunit
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How does the MutSHL?
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The Mut H binds hemimethylated
GATC sequnce. The Mut S will scan for the mismatched base pair. The Mut L will then connect the Mut S and Mut H and this will activate the Mut H. Mut H will make a single strand cut in the unmethylated strand. -Activation of MutH, cleavage of non-methylated 5’ or 3’ of the G An exonuclease trails the complex and digests the ss-DNA tail. The exonuclease recruited is dependent on which side of the mismatch MutH incises the strand – 5’ or 3’. -Depends which way it will travel= 5'-3' or 3'-5' The single-stranded gap created by the exonuclease can then be repaired by DNA Polymerase III (assisted by single-strand binding protein), which uses the other strand as a template, and finally sealed by DNA ligase. Dam methylase then rapidly methylates the daughter strand. |
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Why do ddNTP or 5AZT not function in DNA replication?
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They haven't got 3'OH
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What is the direct product of a circular piece of DNA in replication?
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linear ssDNA
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Just know this for a gene?
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Transcription Unit
Functional unit of heridity Not just protein encoding -Untranslated regions -Structural and other RNAs Not just the transcribed region -Mutations in promoters and other regulatory regions are heritable and can produce different phenotypes |
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Why is a gene not just in the transcribed region?
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Mutations in promoters and other regulatory regions are heritable and can produce different phenotypes
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Describe the structure of RNA polymerase?
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Has two alpha, one beta (has the polymerase activity (catalyzes the synthesis of RNA, which includes chain initiation and elongation).) and another beta subunit (binds to DNA).
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What provides the specificity of the RNA polymerase?
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Sigma subunits provide promoter specificity
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Does the RNA polymerase require a primer?
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Nope plus it is highly processive
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Describe the promoter region in bacteria?
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-35 region this is the TTGACA
-10 region this is the TATAAT sequence (pribnow region) |
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What is a consensus sequence?
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The consensus sequence shows which residues are most abundant in the alignment at each position.
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Why is it good to have the promoters not always turned on and where do you usually see promoters that are locked into the on position?
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This allows modulation of expression – allows changes in transcriptional rate either up or down, and makes them easier to turn off and on
“Maximal” promoters are typically only found in viruses and phages |
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What are the open and closed complex in RNA polymerase?
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Closed
-Initial binding of RNA polymerase complex to dsDNA (-10 and -35 sites are main points of contact) Open -Partial melting of DNA, ~14 nucleotides |
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The sigma factors in RNA polymerase are used for specificity and allows for tight binding between the core enzyme and the DNA. Is the sigma factor used both initiation and elongation?
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Just initiation
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How many sigma factors are there in E. Coli?
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Eight
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Give an example how sigma factors allow a modular regulation of transcription?
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sigmaF controls flagellar genes. In order to make flagella, many copies of the sigmaF protein are made. They bind RNApol. These complexes then bind the promoter(s) upstream of the genes necessary to make flagella. If the cell does not need to make new flagella, sigmaF is not made.
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Link function of these sigma factors:
Sigma38 / SigmaS sigma32: heat shock, sigma54 / sigmaN: sigma24 / sigmaE sigma70 |
sigma70: “housekeeping” genes, expressed in log phase
sigma24 / sigmaE: extracytoplasmic factor – helps maintain outer membrane and cell wall, resistance to heavy metal sigma54 / sigmaN: Nitrogen responsive – increases in uptake mechanisms for N sigma32: heat shock, controls expression of heat shock proteins Sigma38 / SigmaS: stationary phase genes (dps) |
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What usually signals the transition of the initiation to elongation stage?
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Formation of 9+ nt RNA – if the RNA reaches 9 nt, the RNA is long enough to fold back and interact with RNA polymerase. This causes the transition. The transition to the elongation complex usually involves loss of initiation factors (including σ). The elongation complex is much more processive than the initiation complex.
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How is the DNA replication similar to the RNA elongation?
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Uses energy of incoming NTPs to drive reaction
The elongation complex assembles RNA one nucleotide at a time – it cannot read ahead or behind i.e. it doesn’t read codons It has an error rate and associated proofreading activity However, note that mutations in RNA transcripts are not as detrimental as errors in DNA strand RNA polymerase must deal with same issues of supercoiling that face DNA polymerase Topoisomerases relieve supercoiling allowing transcription to continue |
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Give an example how sigma factors allow a modular regulation of transcription?
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sigmaF controls flagellar genes. In order to make flagella, many copies of the sigmaF protein are made. They bind RNApol. These complexes then bind the promoter(s) upstream of the genes necessary to make flagella. If the cell does not need to make new flagella, sigmaF is not made.
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Link function of these sigma factors:
Sigma 70/"Housekeeping genes" Sigma 24/SigmaE Sigma 54/SigmaN Sigma 32/ heat shock Sigma 38/SigmaS |
sigma70: “housekeeping” genes, expressed in log phase
sigma24 / sigmaE: extracytoplasmic factor – helps maintain outer membrane and cell wall, resistance to heavy metal sigma54 / sigmaN: Nitrogen responsive – increases in uptake mechanisms for N sigma32: heat shock, controls expression of heat shock proteins Sigma38 / SigmaS: stationary phase genes (dps) |
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What usually signals the transition of the initiation to elongation stage?
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Formation of 9+ nt RNA – if the RNA reaches 9 nt, the RNA is long enough to fold back and interact with RNA polymerase. This causes the transition. The transition to the elongation complex usually involves loss of initiation factors (including σ). The elongation complex is much more processive than the initiation complex.
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How is the DNA replication similar to the RNA elongation?
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Uses energy of incoming NTPs to drive reaction
The elongation complex assembles RNA one nucleotide at a time – it cannot read ahead or behind i.e. it doesn’t read codons It has an error rate and associated proofreading activity However, note that mutations in RNA transcripts are not as detrimental as errors in DNA strand RNA polymerase must deal with same issues of supercoiling that face DNA polymerase Topoisomerases relieve supercoiling allowing transcription to continue |
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Some RNA:DNA hybridization does occur, probably provides some stability to complex
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Some RNA:DNA hybridization does occur, probably provides some stability to complex
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What is the function of Rho and where does Rho receive its energy?
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Rho functions as a helicase for ds RNA. It will unwind ds regions of RNA, using the energy provided by ATP hydrolysis
Binds nascent RNA and moves in 5 → 3’ direction |
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If polymerase pauses on RNA what can remove the RNA polymerase?
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The Rho
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What are the two proteins used for transcription termination?
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Rho dependent and independent
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Describe how Rho independent termination functions?
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The nascent RNA strand forms a “hairpin” or “stem-loop” structure at 3’ end (UTR or trailer)
The hairpin sequence is usually followed by a short stretch (~6) of U residues This then causes the RNA polymerase to fall off the RNA strand |
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___________ genes may be polycistronic, i.e. encode more than one ORF
so that different polypeptides can be made from a single mRNA |
Bacterial
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What are the three stop codons?
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UAssholeAsshole
UGreatAsshole UAssholeGreat |
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What does RNA polymerase read?
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It transcribes one base at a time, NOT CODONS OR FRAMES
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Bacteria is polycistronic, what is between the open reading frames?
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The ribosome binding sites
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What is the function of Aminoacyl tRNA synthetase?
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This will, using ATP, will add an amino acid to the tRNA at the last tRNA base at the 3'-end. This is the charging of the tRNA.
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Translation-What is the ribosome binding site in both Eukaryotes and Prokaryotes called?
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Shine Dalgarno-Prokaryotes
Kozak Sequence-Eukaryotes |
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What is the consensus sequence of the shine dalgarno sequence?
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AGGAAG and then there is a spacer that precedes the start codon
Complementary to sequence found in 16S RNA (CUUCCU) |
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What is the purpose of the rRNA?
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The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids and to interact with the tRNAs during translation by providing peptidyl transferase activity
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What component of the rRNA provides the peptidyl transferase action?
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The 23s of the large subunit
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What makes up the rRNA?
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50S-23s,5s, and 34 peptodes
30S-16s and 21 peptides |
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What is tRNA charging and what enzyme has this role?
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The aminoacyl tRNA synthetase is the enzyme that does this. This is what adds amino acids to the tRNA. It makes sure the right tRNA is added.
by recognizing the shape of the tRNA molecule as well as the anticodon loop, it places the correct amino acid onto the tRNA, so that it can be added onto the growing peptide chain by ribosome again. if this were not the case, you'd find that you'd run out of tRNA's with amino acids attached very quickly, and translation would thus stop. |
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Describe the steps on translation initiation?
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The tRNA-met will bind to the 30s
30S + tRNAMet releases IF-3, binds mRNA to form 30S Initiation 50S subunit binds to 30S IC to form 70S Initiation Complex |
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Besides AUG-Methionine, what is also used as the initiation for tRNA?
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GUG-Valine
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Two molecules of what are given off during the formation of the 70s initiation complex and what mediates this?
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GTP
Hydrolysis is mediated by Initiation Factor one and two (IF-1 and IF-2). |
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What uses energy from GTP to load a charged tRNA into the A site?
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EF-Tu
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When amino acids are added to the tRNA (when they are initially charged), what sequence on the tRNA are amino acids added to?
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The 3' end of CCA on the tRNA.
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In the transpeptition activity what provides the energy and what controls the transpeptidase activity?
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The energy in the amino-acyl tRNA provides the energy and the 23s in the small subunit will control this activty
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Where are amino acids added on another amino acid in transpeptidation?
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The 3' end
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What provides the translocase activity in translation and requires what?
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Requires GTP and is provided by EF-G.
The factor EF-G catalyzes the translocation of the tRNA and mRNA down the ribosome at the end of each round of polypeptide elongation. |
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What will release the protein from the tRNA?
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peptidyl transferase (23S)
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________ _______ involved in recognition of stop
codon and release of 30S. GTP hydrolysis involved |
Release factors (RF-1,RF-2,RF-3)
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During termination the 30s will quickly re-bound to the IF-3 to prevent what?
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IF-3 binding to 30S prevents
re-association of 30S |
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Why is Inosine used as an anticodon in tRNA?
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Inosine can be used as the anticodon loop of the tRNA to bind to more than one of the possible mRNA codons
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