Exam 2 Flashcards
What was known of DNA before Watson and Crick?
“Heredity factors” were identified by Mendel, known to exist on chromosomes. But were they protein or DNA?
What types of molecules are chromosomes made out of?
Protein and DNA
Explain the transformation experiments, and who performed them?
Frederick Griffith
Describe the experiment that determined that DNA is the hereditary material of phages, and who performed them?
Hershey-Chase Experiment
Labelled protein coat of phages with 35S
Labelled DNA with 32P
Both are radioactive isotopes
Infect E. coli and determine what component was passed into the cell. Answer was: DNA
List three properties hereditary material must have?
- DNA must allow faithful replication during cell division
- DNA must have informational content
- DNA must be able to change/mutate
What base is this? A purine or pyrimidine?
Adenine
Purine
What base is this? A purine or pyrimidine?
Thymine
Pyrimidine
What base is this? A purine or pyrimidine?
Guanine
Purine
What base is this? A purine or pyrimidine?
Uracil
Pyrimidine
What base is this? A purine or pyrimidine?
Cytosine
Pyrimidine
Chargaff’s Rule
purines = # pyrimidines
A+G = T+C
A=T and G=C
Suggests a purine must pair with a pyrmidine, and base pairs must be restricted to A-T and G-C
GC Content
A+T does not equal G+C
Amount of GC/AT varies across organisms
What did X-ray crystallography discover about DNA?
A purine + a pyrimidine is the correct thickness for DNA
What is one “bead” of a nucleosome composed of?
8 histone protein molecules + 146 bp of DNA, connected by linker DNA
What experiment determined the manner in which DNA is replicated? Who performed it?
Meselson-Stahl Experiment
Used 15N and 14N labels to differentially weight DNA, if replication was not conservative, new replicants would show up in different locations after centrifugation. They did not.
What are the three models of DNA replication predicted by Meselson-Stahl?
Semiconservative: new DNA contains a parental strand and a newly sythesised strand (correct).
Conservative: new DNA is either all parental or all newly synthesised
Dispersive: new DNA contains random bits of parental material scattered throughout
What experiment determined that replication occurs at a replication fork?
In his 1963 paper “The bacterial chromosome and its manner of replication as seen by autoradiography”, John Cairns demonstrated by autoradiography that the DNA of the bacterium Escherichia coli was a single molecule that is replicated at a moving locus (the replicating fork) at which both new DNA strands are being synthesized. Subsequently, it was found that there were in fact two moving forks, traveling simultaneously in opposite directions around the chromosome. He used a pyrimidine deoxynucleoside, thymidine, radioactively labelled with 3H. Intially grew cells in small amounts of 3H-thymidine, then breifly in large amounts of 3H-thymidine. A dense label at the replication fork as new DNA is made
Requirements for DNA Replication
- Single-stranded DNA so nucleotide bases can pair = Enzyme Helicase
- A primer – must provide a free 3’ OH group for the 5’ end of another nucleotide to bind = Enzymes Primase
- All four free nucleotides (ie. A, T, C, G) available in the nucleus = dNTPs
- Polymerase – enzyme that catalyzes addition of nucleotides = DNA polymerase 3 (pol III) and DNA polymerase 1 (pol I)
At the replication fork, what end of paternal DNA is the leading strand? The lagging strand? What order are new dNTPs added?
The leading strand has a parental 3’ end, while the lagging strand has a parental 5’ end. New dNTPs are always added from 5’ to 3’!
DNA helicase
Unzips dsDNA by breaking hydrogen bonds between bases
DNA primase
Places RNA primers (with a free 3’ OH) onto the “naked” template strand so that DNA polymerase III can add new dNTPs in the 3’ direction
Does DNA primase work on the leading or the lagging strand?
BOTH! Only one RNA primer is required on the leading strand, many required on the lagging strand
DNA polymerase forms what type of bond?
A phosphodiester bond between 3’ OH and 5’ phosphate of dNTPs
DNA polymerase I
DNA polymerase I (pol I)
- Polymerase activity – 5’ to 3’ activity
- A 3’ to 5’ exonuclease activity to remove mismatched bases
- A 5’ to 3’ exonuclease activity which degrades single strands of DNA or RNA – removes RNA primers set down for replication
DNA polymerase II
DNA polymerase II (pol II)
•A 3’ to 5’ exonuclease activity to remove mismatched bases
DNA polymerase III
DNA polymerase III (pol III)
Primary enzyme involved in forming phosphodiester bond that extends growing chain
•Polymerase activity – 5’ to 3’ activity
What DNA polymerases have a 3’ to 5’ exonuclease activity?
Pol I and Pol II
3’ to 5’ exonuclease activity removes mismatched bases
What DNA polymerases have 5’ to 3’ polymerase activity?
Pol I and Pol III
Pol III is the primary enzyme involved in forming new phosphodiester bonds that extend DNA
Which DNA polymerase operates first, Pol I or Pol III?
DNA polymerase III first extends new DNA from RNA primers, then Pol I removes the RNA primers and repairs with DNA
DNA ligase
Joins Okazaki fragments
The E. coli DNA ligase is encoded by the lig gene. DNA ligase in E. coli, as well as most prokaryotes, uses energy gained by cleaving nicotinamide adenine dinucleotide (NAD) to create the phosphodiester bond. It does not ligate blunt-ended DNA except under conditions of molecular crowding with polyethylene glycol, and cannot join RNA to DNA efficiently.
What is the name for the origin of replication in prokaryotes?
oriC
DnaA
DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication initiation factor which promotes the unwinding of DNA at oriC. The onset of the initiation phase of DNA replication is determined by the concentration of DnaA
What sequences are found at the origin of replication in prokaryotes?
An AT-rich sequence where DNA is unwound, and DnaA boxes with concensus sequence TTATTNCACA (TTATCCACA for exam purposes)
DnaA boxes
The DnaA protein is an essential factor for initiation of duplication of the bacterial chromosome from a specific site, the origin of replication, oriC. The DnaA protein binds to DnaA boxes in oriC, and in vitro studies indicate that initiation takes place when sufficient DnaA protein—approximately 20 monomers—has been bound to oriC. The formation of this so-called initial complex leads to the opening of a region in oriC containing AT-rich 13-mers and allows the entry of DnaB and DnaC proteins to form the pre-prepriming complex, which is followed by several other stages. The DnaA protein binding sites—the DnaA boxes—have the consensus sequence TTATTNCACA
Describe the replisome
A coordinated multiprotein complex, about 1 kb/second in E. coli.
DNA gyrase
DNA gyrase (a topoisomerase) removes extra twists
What makes eukaryotic DNA replication more complex than in prokaryotes?
The presence of nucleosomes
How many replisomes are present at each replication fork?
Two, DNA replication is bidirectional
At what stage of the cell cycle do cells divide? Does DNA replicate?
Cells divide during M (mitosis) phase, DNA replicates during S (DNA synthesis) phase
How is DNA replication in eukaryotes restricted to S phase?
Cdc6 and Cdt1 are synthesized just before S phase and destroyed afterward to restrict DNA replication to S phase.
What two proteins are required for eukaryotic DNA replication initiation?
Cdc6 and Cdt1
What end of DNA does telomerase attatch to?
Telomerase attatches to the 3’ end to extend the 5’ end, because DNA polymerase can only extend in the 5’ to 3’ direction
What is telomerase made of?
Telomerase is an enzyme that contains both an RNA and a protein component. The template for the polymerase activity of the enzyme is a small RNA that is an integral part of the enzyme.
Telomeric repeats
TTAGGG sequenes (repeated thousands of times at the end of human chromosomes, for example)
Who proposed the RNA world hypothesis, in what year?
Francis Crick in 1968
tRNA
Transfer RNA (tRNA) – brings correct amino acid to mRNA
rRNA
Ribosomal RNA (rRNA) – major component of ribosomes
Three main differences between RNA and DNA
Ribose in RNA, deoxyribose in DNA
Uracil in RNA, 5-methyluracil (thymine) in DNA
Single strand in RNA, usually double strands in DNA
Describe the genetic material of ebola
Ebola – A single stranded negative sense RNA virus
What direction is DNA transcribed in?
Always from 3’ to 5’
New RNA, like DNA, is added to the free 3’ OH
Only one strand of DNA is the template for any gene transcription, but which strand varies with the gene. Genes transcribed in different directions use opposite strands of the DNA as templates
What end is exposed of the nascent mRNA transcript?
The 5’ end (new dNTPs are added in the ribosome, so the 5’ end must exit the ribosome first)
Transcription, like replication, is always 5’ to 3’
When referring to a gene (in DNA) what strand do we describe? What is the convention for writing it?
Gene orientation always refers to the non-template strand (or sense strand), thus genes are always written in the same direction that RNA is transcribed 5’ à 3’ direction
What direction is “upstream” of the promoter? “Downstream”?
5’ is upstream
3’ is downstream
mRNAs are synthesised in the 5’ to 3’ direction
Where is the start codon in reference to the promoter?
The start codon (AUG) appears after a 5’ UTR (untranslated region)
What does the promoter sequence contain in prokaryotes?
The -35 box: TTGACAT
The -10 box (Shine-Dalgarno sequence): TATAAT
Describe the subunits of prokaryotic RNA polymerase
2 α – interact regulatory proteins
1 β – catalysis
1 β’ – binds DNA
1 ω – enzyme assembly/regulation
Sigma (σ) factors – similar to transcription factors in Eukaryotes bind and recognize the -35/-10 consensus motifs
Prokaryotic RNA polymerase α subunit
Interacts with regulatory proteins
Prokaryotic RNA polymerase β subunit
Catalysis
Prokaryotic RNA polymerase β’ subunit
Binds DNA
Prokaryotic RNA polymerase ω subunit
Enzyme assembly/regulation
Prokaryotic RNA polymerase σ factor
Similar to transcription factors in Eukaryotes bind and recognize the -35/-10 consensus motifs
What is the sequence of the start codon in DNA?
ATG
(AUG once translated to RNA)
What is the sequence of the stop codon in DNA?
TAA, TAG, TGA
(UAA, UAG, UGA once transcribed to RNA)
In prokaryotes, is the entire mRNA transcript transcribed?
No, there is a 5’ UTR and a 3’ UTR
Intrinsic transcript termination
Also called Rho-independent termination
In the intrinsic mechanism, the termination is direct. The terminator sequences contain about 40 base pairs, ending in a GC-rich stretch that is followed by a string of six or more A’s. Because G and C in the template will give C and G, respectively, in the transcript, the RNA in this region also is GC rich. These C and G bases are able to form complementary hydrogen bonds with each other, resulting in a hairpin loop (shown). Recall that the G–C base pair is more stable than the A–T pair because it is hydrogen bonded at three sites, whereas the A–T (or A–U) pair is held together by only two hydrogen bonds. Hairpin loops that are largely G–C pairs are more stable than loops that are largely A–U pairs. The loop is followed by a string of about eight U’s that correspond to the A residues on the DNA template.
Normally, in the course of transcription elongation, RNA polymerase will pause if the short DNA–RNA hybrid in the transcription bubble is weak and will backtrack to stabilize the hybrid. Like that of hairpins, the strength of the hybrid is determined by the relative number of G–C base pairs compared with A–U base pairs (or A–T base pairs in RNA–DNA hybrids). In the intrinsic mechanism, the polymerase is believed to pause after synthesizing the U’s (which form a weak DNA–RNA hybrid). However, the backtracking polymerase encounters the hairpin loop. This roadblock sets off the release of RNA from the polymerase and the release of the polymerase from the DNA template.
Rho-dependent termination
RNAs with rho-dependent termination signals do not have the string of U residues at their 3’ end and usually do not have hairpin loops (as in intrinsic termination). Instead, they have a sequence of about 40 to 60 nucleotides that is rich in C residues and poor in G residues and includes an upstream segment called the rut (rho utilization) site. Rho is a hexamer consisting of six identical subunits that bind a nascent RNA chain at the rut site. These sites are located just up- stream from (recall that upstream means 5 of) sequences at which the RNA polymerase tends to pause. After binding, rho facilitates the release of the RNA from RNA polymerase. Thus, rho-dependent termination entails the binding of rho to rut, the pausing of polymerase, and rho-mediated dissociation of the RNA from the RNA polymerase.
rut
Rho utilisation site: the site on the mRNA that Rho binds to
Rho-dependent termination entails the upstream binding of Rho to rut, the pausing of polymerase, and rho-mediated dissociation of the RNA from the RNA polymerase.
In eukaryotic transcription, how does the pre-initation complex assemble?
TATA binding protein (TBP) that is part of TFIID first binds to the TATA box, then other TFII’s and finally RNA polymerase bind to the pre-initiation complex
What event starts eukaryotic transcription?
The carboxy tail domain (CTD) is phosphorylated by general transcription factors (GTFs), RNA polymerase II begins elongation
