Exam 2 Flashcards

Question

DNA polymerase II

Answer 1

DNA polymerase II (pol II) •A 3’ to 5’ exonuclease activity to remove mismatched bases

Answer 2

DNA polymerase III (pol III) **Primary enzyme involved in forming phosphodiester bond that extends growing chain** •Polymerase activity – 5’ to 3’ activity

Answer 3

Pol I and Pol II 3' to 5' exonuclease activity removes mismatched bases

Answer 4

Pol I and Pol III Pol III is the primary enzyme involved in forming new phosphodiester bonds that extend DNA

Answer 5

DNA polymerase III first extends new DNA from RNA primers, then Pol I removes the RNA primers and repairs with DNA

Answer 6

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.

Answer 7

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

Answer 8

An AT-rich sequence where DNA is unwound, and DnaA boxes with concensus sequence TT^A_TTNCACA (TTATCCACA for exam purposes)

Answer 9

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 **TT^A_TTNCACA**

Answer 10

A coordinated multiprotein complex, about 1 kb/second in *E. coli*.

Answer 11

DNA gyrase (a topoisomerase) removes extra twists

Answer 12

The presence of nucleosomes

Answer 13

Two, DNA replication is bidirectional

Answer 14

Cells divide during M (mitosis) phase, DNA replicates during S (DNA synthesis) phase

Answer 15

Cdc6 and Cdt1 are synthesized just before S phase and destroyed afterward to restrict DNA replication to S phase.

Answer 16

Cdc6 and Cdt1

Answer 17

Telomerase, also called telomere terminal transferase, is a ribonucleoprotein that adds the polynucleotide "TTAGGG" to the 3' end of telomeres, which are found at the ends of eukaryotic chromosomes. A telomere is a region of repetitive sequences at each end of a chromatid, which protects the end of the chromosome from deterioration or from fusion with neighbouring chromosomes. Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule (with the pattern of "CCCAAUCCC" in vertebrates), which is used as a template when it elongates telomeres.

Answer 18

Found in *Tetrahymena* by Carol Greider and Elizabeth Blackburn. Mixed a cell extract with synthetic telomere DNA oligomers (as primers) and dNTPs, found telomere DNA primer extended.

Answer 19

Telomerase attatches to the 3' end to extend the 5' end, because DNA polymerase can only extend in the 5' to 3' direction

Answer 20

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.

Answer 21

Briefly, the telomerase RNA first anneals to the 3' DNA overhang (**telomerase engagement**), which is then extended with the use of the telomerase’s two components: the small RNA (as template) and the protein (as polymerase activity, **reverse transcription**). After the addition of a few nucleotides to the 3' overhang, the telomerase RNA moves along the DNA so that the 3' end can be further extended by its polymerase activity. The 3' end continues to be extended by repeated movement of the telomerase RNA. Primase and DNA polymerase then use the very long 3' overhang as a template to fill in the end of the other DNA strand (**DNA polymerase elongation**).

Answer 22

TTAGGG sequenes (repeated thousands of times at the end of human chromosomes, for example)

Answer 23

The cap structure at the end of each chromosome is a complex of the ssDNA at the 3’ end and specific proteins. It serves (among many other functions) to protect the ends of the chromosomes from double-strand break repair enzymes.

Answer 24

Causes premature aging Due to a mutation in WRN1m a gene that encodes a protein involved in telomere cap structure

Answer 25

It is increased in 80-90% of cancers, allowing the cells to divide indefinately

Answer 26

Francis Crick in 1968

Answer 27

Messenger RNA, the template for translation

Answer 28

Transfer RNA (tRNA) – brings correct amino acid to mRNA

Answer 29

Ribosomal RNA (rRNA) – major component of ribosomes

Answer 30

Small nuclear RNA (snRNAs) – part of spliceosome

Answer 31

MicroRNA (miRNA) – regulate translation of mRNA

Answer 32

Small interferring RNA/piwi-interacting (piRNA) – genome defense, helps prevent spread of transposable elements

Answer 33

Long noncoding RNAs (lncRNA) – dosage response

Answer 34

Ribose in RNA, deoxyribose in DNA Uracil in RNA, 5-methyluracil (thymine) in DNA Single strand in RNA, usually double strands in DNA

Answer 35

Ebola – A single stranded negative sense RNA virus

Answer 36

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

Answer 37

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'

Answer 38

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

Answer 39

5' is upstream 3' is downstream mRNAs are synthesised in the 5' to 3' direction

Answer 40

* Initiation – Recognition of start site, recruitment of RNA polymerase * Elongation – RNA polymerase synthesizes mRNA 5’ à 3’ * Termination – RNA polymerase is kicked off of DNA template strand by either instrinsic (hairpin loop) or Rho-dependent mechanisms

Answer 41

The start codon (AUG) appears after a 5' UTR (untranslated region)

Answer 42

The -35 box: TTGACAT The -10 box (Shine-Dalgarno sequence): TATAAT

Answer 43

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

Answer 44

Interacts with regulatory proteins

Answer 45

Enzyme assembly/regulation

Answer 46

Similar to transcription factors in Eukaryotes bind and recognize the -35/-10 consensus motifs

Answer 47

σ factors dissociate and RNA polymerase moves down the DNA

Answer 48

ATG | (AUG once translated to RNA)

Answer 49

TAA, TAG, TGA | (UAA, UAG, UGA once transcribed to RNA)

Answer 50

No, there is a 5' UTR and a 3' UTR

Answer 51

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.

Answer 52

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* (*r*ho *ut*ilization) 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.

Answer 53

*R*ho *ut*ilisation 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.

Answer 54

RNA polymerase II

Answer 55

They transcribe rRNA, tRNA and some snRNA * **RNA polymerase I** – transcribes rRNA except 5.8S subunit * RNA polymerase II – transcribes all protein-coding mRNA and some snRNA * **RNA polymerase III** – transcribes small functional RNA genes (tRNA, 5.8S, some snRNA)

Answer 56

TATA box is eukaryotic and -30 Shine-Dalgarno sequence is prokaryotic and -10

Answer 57

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

Answer 58

The carboxy tail domain (CTD) is phosphorylated by general transcription factors (GTFs), RNA polymerase II begins elongation

Exam 2 Flashcards

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