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Describe the general structure of a eukaryotic DNA molecule.

-A linear polymer made up of nitrogenous bases (nucleotides) adenine, guanine, cytosine, thymine
-Takes the shape of a double helix
-Has polarity, a 3' and a 5' end
-Each strand bound together by covalent bonds, and the strands are bonded together by H-bonds
-Nucleotides are always added to the free 3' end


Describe the general steps in DNA replication.

1) Helicase opens helix
2) Topoisomerase relieves tension by nicking to untwist
3) RNA primase synthesizes a primer at the 3' end of the leading strand, and along the lagging strand as it is unravelled.
4) Pol III adds nucleotides continuously to the leading strand, and fills in the RNA primers on the lagging strand.
5) Pol I excises RNA primers (5' - 3' exonuclease activity) and fills in gaps.
6) Ligase bonds the Okazaki fragments together.


What are the functions of Pol I?

1) 3' - 5' exonuclease - proofreading
2) 5' - 3' exonuclease - removes RNA primers
3) 5' - 3' synthase/polymerase


What is the major elongation enzyme?



What is the overhang problem?

It is impossible to replicate the very end of the lagging strand, because once the last 5' - 3' RNA primer is removed, there is no free 3' end for pol III to add nucleotides to. This leads to the shortening of DNA after each replication.


How do telomeres and telomerase solve the overhang problem?

Telomeres are the regions on the ends of our DNA molecules made up of mostly repeats - they do not code for anything. This protects us from losing coding information during DNA replication.

Telomerase is a ribonuclear protein containing an RNA template. It binds to the 3' overhang and extends it further. An RNA primase then comes and synthesizes another primer, and DNA pol III synthesizes complementary strand. There is still an overhang, but telomeres are continuously lengthened to prevent fast degradation.


List the enzymes and proteins involved in DNA replication and their functions.

Primase - synthesizes RNA primers based on template strand
Pol III - elongates RNA primers with nucleotides
Pol I - removes RNA primers and fills gaps; proofreads
Helicase - unzips DNA H-bonds
Topoisomerase (gyrase)- nicks DNA to untwist
SSBPs - bind to single-strand DNA to prevent duplex from reforming
ß-Clamp - encircles DNA like a donut and keeps pol III attached
Ligase - forms covalent bonds between okazaki fragments on lagging strand


What are the structural differences between DNA and RNA?

1) RNA molecules have a hydroxyl group at the 2’ position of the sugars that DNA molecules lack.
2) RNA molecules are less stable and turnover more rapidly than DNA molecules.
3) RNA molecules have U in place of T found in DNA molecules.
4) Some RNA molecules can catalyze biological reactions, but DNA cannot.


Why does each eukaryotic chromosome contain many origins of replication?

Eukaryotic genomes are much larger, so more origins are required to replicate more quickly.


Describe the proposed models for DNA replication. Which one was correct?

Conservative - the two parental DNA strands are back together after replication has occurred.
Semiconservative - the two parental DNA strands separate and each of those strands then serves as a template for the synthesis of a new DNA strand (*CORRECT*)
Dispersive - parental double helix is broken into double-stranded DNA segments that act as templates for the synthesis of the new double helix molecules.


Why are G-C pairs stronger than A-T pairs?

They have one more hydrogen bond that A-T pairs.


What are some similarities between DNA replication and DNA transcription?

1) Use similar nucleotide building blocks
2) Use same chemical method of attach by a terminal hydroxyl group of growing chain on the triphosphate group of an incoming nucleotide with the use of an RNA polymerase.
3) Start in a bubble.
4) Synthesize in a 5' to 3' direction.


How is DNA transcription different from DNA replication?

1) DNA transcription generates a single-stranded mRNA
2) Only one DNA strand is used as a template
3) No supercoils (bubble never long enough), so topoisomerase is not needed
4) RNA polymerase can act without a primer
5) Does not transcribe the entire chromosome at once


Describe the steps of DNA transcription in prokaryotes.

1. RNA polymerase complexed with a sigma factor binds to promoter "consensus DNA sequence"
2. Double-stranded DNA goes through a conformational change to form an open complex through opening of the base pairs at the -10 position.
3. Initiator nucleotide binds to complex. First phosphodiester bonds are made.

1. In transcription bubble, double helix is unwound and RNA polymerase monitors the binding of a free ribonucleoside triphosphate to the next exposed base on the DNA template strand.
2. DNA that is unwound ahead of RNA polymerase is rewound after it has been transcribed.

1. RNA synthesis will proceed until RNA polymerase recognizes special nucleotide sequences that act as a signal for chain termination.
a. Rho-independent (simple termination)
- Formation of a hairpin loop in GC rich regions of template renders the strand inaccessible to RNA polymerase.
b. Rho-dependent
- A protein factor (Rho) binds to the termination site and destabilizes the interaction between DNA template and RNA polymerase. The mRNA is released.


Describe the steps of DNA transcription in eukaryotes.

1. GTFs mediate the binding of RNA polymerase to the promoter region. TFs are required for proper assembly of the transcription-initiation complex.
a. Conserved sequences: TATA box ~ -25,
2. DNA double helix is opened up.

1. RNA polymerase begins adding nucleotides to the growing transcript, catalyzing the formation of phosphodiester bonds between them.
2. As the strand of RNA is synthesized, the already-transcribed regions of DNA rewind together and the mRNA is dissociated.

1. Occurs when RNA polymerase reaches a stop sequence—often, a polyadenylation signal (AAUAAA).


What occurs during pre-mRNA processing?

1) Processing of 3' and 5' ends
- addition of 5' cap: necessary for translation, and protective
-addition of poly-A tail: a stretch of ~200 adenine nucleotides is added; length determines how long RNA will live in the cytoplasm
2) Splicing: removal of introns


What is the mechanism of splicing?

1) snRNPs bind to 5' splice site and internal A site
2) other snRNPs join the spliceosome
3) First splicing reaction; one intron end attaches to A
4) Second splicing reaction; other intron end cleaved
5) Exons join


What are the different classes of RNA?

-Messenger RNA: carries genetic information from the DNA in the nucleus to ribosomes in the cytoplasm (sites of protein synthesis); 5’ cap (also involved in recognition of mRNA by translating machinery) and 3’ poly(A) tail greatly enhance the stability of mRNA molecules

-Functional RNA: does not encode information to make a protein, as the RNA itself is the final functional product.
♣ Transfer RNA: brings the correct amino acid from the cytoplasm to the mRNA during translation.
♣ Ribosomal RNA: major component of ribosomes (guide the assembly of the amino acid chain). May act as ribozymes.
♣ Small nuclear RNA: found in eukaryotic nucleus, assists in processing of RNA transcripts, may unite with protein subunits to form the spliceosome.
♣ MicroRNA: regulate the amount of protein produced by many eukaryotic genes. Exert their regulatory action by binding messenger RNAs and preventing their translation into proteins.
♣ siRNA: double stranded RNA that interferes with expression of a specific gene by hybridizing to its corresponding RNA sequence in the target mRNA, activating the degrading mRNA. Once the target mRNA is degraded, it can no longer be translated.


Differentiate between the sense and antisense strands.

-Sense strand: also known as the “coding strand,” refers to the strand of DNA running from 5’ to 3’. Has the same sequence as the mRNA. Is not used by the cell to make protein.

-Antisense strand: also known as the ”template strand,” refers to the strand that is used as the source for the protein code. Complementary to the mRNA.


Where does energy for DNA replication come from?

the breaking of the triphosphate group on a nucleotide releases energy used to form covalent bond


What is the purpose of alternative splicing?

Different mRNAs and therefore different proteins are produced by splicing exons together in different combinations.


How is miRNA processed and what is it's function?

1) Transcribed as a longer RNA in the form of a double-stranded stem with a loop, and a mismatched base pair on the stem
2) Processed in the nucleus to a smaller but not final form
3) In cytoplasm, binds to Dicer, which cleaves them into 22 nt products
4) RISC binds to short double stranded RNA and unwinds it into single-stranded miRNA
5) miRNA (still bound to RISC) binds to complimentary mRNAs, and represses translation or removes poly-A tail

The function is to regulate expression of genes.


How is siRNA different from miRNA?

-Instead of regulating other genes, siRNA silences the gene that produces it.
-function is to safeguard the genome from viruses and transposons

1) Antisense RNA is formed in response to the insertion of foreign DNA in the genome
2) DNA detects the double-stranded RNA that forms between antisense and sense RNA and processes it into short RNAs
3) RISC binds a short RNA and unwinds it to form siRNA
4) siRNA targets RISC to a perfectly complementary mRNA, which is degraded
5) Silences the foreign gene


What is the result of inserting either a dsRNA copy of a gene, or the gene itself, into embryos?

Transgene insertion leads to the synthesis of antisense RNA, which complements with sense RNA to form dsRNA. Therefore, in both instances, siRNA is formed and silences the expression of the gene in question.


List all of the nucleic acid signals you can think of.

1. Promoter (TATA)
2. Origins of Replication / Termination sequence (transcription)
3. Splicing Signals
4. Inverted Repeats (acted upon by transposase)
5. Centromeres (part of chromosome that attaches to spindle proteins)


How is the structure of tRNA adaptive for its function?

tRNA has an anticodon which binds to the codon on the mRNA that specifies the amino acid the tRNA is carrying


Where does the energy for translation come from?

The energy for the ribosome movement, and keeping the tRNA close together comes from the hydrolysis of GTP attached to elongation factors.

The energy for the formation of covalent bonds comes from the breaking of the peptide bond between the "charged" tRNA and its attached amino acid.


What end of the polypeptide is synthesized first?

The N-terminus end is synthesized first, and new amino acids are added to the C-terminus end.


What is wobble?

Some tRNAs can bring their specific amino acids to any one of several codons through loose base-pairing at the 3' end of the codon and the 5' end of the anticodon.


What are the sites of the ribosome?

A site - binds an incoming aminoacyl tRNA
P site - the tRNA in this site binds the growing polypeptide chain
E site - contains a deacylated tRNA ready for release