BCEM 341 - Nucleic Acids

DNA is _____________ into RNA, and _____________ into protein.

transcribed, translated

Nucleic acids (DNA and RNA) are the _______________ in the cell.

informational macromolecules

DNA and RNA are _________, and the carriers of ______________.

polymers, genetic information

Each repeating unit of DNA and RNA has what 3 components?

sugar, phosphate + base

What is the difference between the sugars in DNA and RNA?

DNA → deoxyribonucleic acid (-H on carbon 2)

RNA → ribonucleic acid (less stable, susceptible to hydrolysis because of the -OH group on carbon 2)

Sugar-phosphate backbones are formed through ______________.

phosphodiester bonds

What is a phosphodiester bond?

a bond that links the 3’ C on one nucleotide to the 5’ C on an adjacent nucleotide, via a phosphate

What is a nucleotide?

the repeating unit in nucleic acids (base, sugar, phosphate)

What are the 2 types of bases found in nucleic acids?

purines (double ring structure)

pyrimidines (single ring structure)

What type of bases are adenine and guanine?

purines

What type of bases are cytosine, thymine and uracil?

pyrimidines

What is the most important difference between uracil and thymine?

uracil → RNA only

thymine → DNA only

What is the difference between a nucleoside and a nucleotide?

nucleoside = base bonded to a sugar

nucleotide = nucleoside + one or more phosphate groups

Nucleic acid sequences are always written in the _____________direction.

5’ to 3’

Which end of a nucleic acid polymer has an -OH group that is attached to a phosphate group?

the 5’ end

Which end of a nucleic acid has a “free” -OH group?

the 3’ end (-OH group on carbon 3)

What is the central dogma in molecular biology?

DNA creates proteins via, transcription, translation, and replication

General features of DNA structure?

• Two strands in an ______________ orientation form a ___________________.

• The __________________ is on the outside of the helix, and the _______________ face the middle.

• Bases are ______________ to the helical axis

• Bases are separated by ________.

• The helix is approximately ________ wide.

anti-parallel, right-handed helix

sugar-phosphate backbone, purine and pyrimidine bases

perpendicular

3.4 A

20 A

Guanine pairs with __________.

Adenine pairs with __________ and __________.

cytosine (C)

thymine (T, DNA) and uracil (U, RNA)

How many hydrogen bonds does the pairing of guanine and cytosine have?

3 (more stable)

How many hydrogen bonds does the pairing of adenine with thymine or uracil have?

2

What is semi-conservative replication?

as DNA is replicated, one of the strands in the new daughter DNA molecule comes from the original parent and one is newly synthesized

What was the experiment that Meselson and Stahl performed?

an experiment to prove that DNA replication is semi-conservative by labelling parent DNA with N15 (heavier nitrogen isotope) and transferring cells to new medium containing only N14 (ordinary, lighter nitrogen). More rounds of replication resulted in more N14 and less N15

What is a prokaryote?

an organism lacking a nucleus and membrane-bound compartments (organelles)

• single, circular, double-stranded DNA

• includes bacteria

What is a eukaryote?

an organism whose cells contain a nucleus and other membrane-bound compartments (organelles)

• DNA is in multiple linear molecules (chromosomes)

• includes plants, animals, fungi

Bacterial chromosomes are ____________. To help fit inside a cell, the double helix twists on itself to form a __________.

circular, supercoil

How many pairs of chromosomes do humans have?

23 different chromosomes (2 of each)

What are chromosomes?

DNA protein complexes that compact DNA in eukaryotes

What are nucleosomes?

complexes of DNA and histone proteins (basic, positively charged proteins) linked together that appear like beads on a string

What is chromatin?

general term for the DNA and associated proteins that make up chromosomes

Nucleosomes are arranged in _______ fibers. Compaction of the fibers generates the condensed __________.

30 nm, chromosome

DNA polymerases promote the formation of ____________________ between incoming ________________ and an existing DNA strand.

phosphodiester linkages, deoxyribonucleotide triphosphates (dNTPs)

Which polymerase is the main enzyme that adds nucleotides during DNA replication?

DNA polymerase III

What are the main characteristics of DNA synthesis? (4)

1. the reaction requires all 4 dNTPs: dATP, dGTP, dCTP, and dTTP

2. newly synthesized DNA uses existing DNA as a template

3. DNA polymerase requires a primer to begin synthesis

4. many DNA polymerases can correct errors by removing incorrect nucleotides

Is ATP required for DNA synthesis?

NO

In which direction is DNA synthesized?

in the 5’ to 3’ direction

What occurs at the 3’ OH of the growing strand during synthesis?

nucleophilic attack on the alpha phosphate of the incoming nucleotide

In DNA synthesis, the incoming nucleotide can only be added onto the __________.

3’ end

What is the structure of DNA polymerase?

resembles a right hand (see lecture 27, pg. 11)

• "“fingers + thumb” wrap around DNA + keep it close to the active site

• the active site is in the “palm” domain

When a dNTP binds the active site of DNA polymerase, what happens?

conformation change: the “fingers” rotate to form a pocket in which only the correct match will fit

DNA pol I and DNA pol III both have ___________ activity, which is the ability to remove mismatched nucleotides from the 3’ end of DNA.

3’ to 5’ exonuclease

Proofreading increases replication accuracy by ___________.

1000-fold

DNA _________acts like a wedge to separate strands and allow each strand to act as a ___________ for synthesis.

helicase, template

What is required for DNA synthesis to begin?

an RNA primer, because DNA polymerase requires a 3’ OH on an existing chain to add nucleotides

Primase (an ______________) synthesizes a __________, which is a short stretch of RNA (~10 nucleotides) complementary to the template strand. It DOESN’T require ___________________ to polymerize nucleotides.

RNA polymerase, primer, an existing 3’ OH

What is the site of DNA synthesis?

replication fork

Why is one strand of DNA synthesized continuously and the other in fragments? What are the fragments called?

because synthesis can’t occur in the 3’ to 5’ direction, Okazaki fragments

What are the leading and lagging strands?

leading strand: synthesized continuously

lagging strand: synthesized in Okazaki fragments

DNA pol I has 5’ to 3’ exonuclease activity to remove the _____________. The gap is then filled with DNA by _____________.

RNA primer, DNA pol I polymerase activity

What does DNA ligase do?

catalyzes phosphodiester bonds between Okazaki fragments (uses ATP)

What is PCR?

polymerase chain reaction, a method to synthesize millions of copies of a particular DNA sequence in vitro

What are the required components for PCR?

1. Template DNA: contains the DNA sequence that we want to amplify

2. dNTPs (lots of each): dATP, dGTP, dCTP, dTTP

3. DNA polymerase

4. 2 DNA primers (lots of each):

   • DNA sequence approximately 20 nucleotides long with a free 3’ -OH group

   • 1 primer binds to each strand of the double helix

What are the steps of PCR (steps 1-3 = 1 cycle)?

1. strand separation: 2 strands of the template DNA are separated (heating at 95 degrees C)

2. Hybridization of primers: cool to 54 degrees C to allow primers to anneal (hybridize/bind) to DNA strands

3. DNA synthesis: 72 degrees C is the optimal temperature for the DNA polymerase used in PCR, polymerase elongates both primers in the 5’ to 3’ direction

Why does the DNA sequence get amplified exponentially with PCR? What is the formula to find the amplification of the DNA sequence?

each new strand that is made can act as a template in the next cycle

2^n

What are the 4 types of DNA damage?

• mismatch

• insertion

• deletion

• breaks

What is a mismatch error?

errors in the replication process that results in incorrect base pairing

What is an insertion error?

insertion of one or more additional base pair(s) into the sequence

What is a deletion error?

deletion of one or more base pair(s) from the sequence

What is a break error?

break in one or both strands of the double helix that can cause DNA polymerase to stall or fall off during DNA replication

What is a mutagen? What kind of DNA damage does a mutagen cause?

chemical agent that can alter specific bases within DNA, results in an overall mismatch

What is deamination? How can it be a mutagenic process?

removal of amine group, can be a mutagenic process because it alters adenine into hypoxanthine which pairs with cytosine (mismatch)

What kind of error will epoxides cause?

mismatch

What are the 3 general steps of DNA repair mechanisms?

1. recognize the offending base(s)

2. remove the offending base(s)

3. repair the resulting gap in DNA

DNA pol I and DNA pol III both have 3’ to 5’ exonuclease activity (ability to remove mismatched nucleotides from the 3’ end of DNA). How does this occur?

mismatched strand causing pausing by the polymerase and mismatched strand is likely to flop around + interact with the exonuclease site

What is the function of the mismatch repair?

repairs errors that were not corrected by DNA polymerase’s proofreading

How does mismatch repair function?

1. recognition of mismatch: MutS or MutL detects mismatch

2. removal: MutH endonuclease makes an internal cut + exonuclease removes part of the strand containing the error

3. repair: gap is filled by DNA polymerase + sealed by DNA ligase

What is a plasmid?

small, circular, double-stranded DNA molecule (separate from the chromosomal DNA) that can replicate independently

DNA is ___________charged, so it migrates to the ___________ end of the gel. Smaller DNA migrates through the gel more ___________ than larger DNA.

negatively, positive, quickly

What are the 3 different conformations of plasmids? What order will they be found in on a gel electrophoresis?

• supercoiled (smallest)

• circular relaxed (largest)

• linear

from farthest to closest: supercoiled, linear, circular relaxed

What are restriction enzymes (restriction endonucleases)?

enzymes that recognize specific DNA sequences + cleave at specific sites on both strands of the DNA double helix

The sequences recognized by restriction enzymes are _______________.

palindromic

RNA is unstable and is _____________ relatively quickly.

degraded

RNA is synthesized by ______________.

RNA polymerases

What is RNA polymerase? What is its function?

complex enzyme containing multiple subunits which promotes the formation of phosphodiester linkages between incoming ribonucleotides (rNTPs)

What are the 3 main characteristics of RNA synthesis?

1. reaction requires all 4 ribonucleotides: ATP, GTP, CTP, and UTP

2. RNA synthesis uses DNA as a template

3. RNA polymerase does NOT require a primer to begin synthesis

How does the mRNA sequence relate to the coding and template strands of DNA?

mRNA is the same as the coding strand of DNA, but just has U’s instead of T’s (both 5’ to 3’)

mRNA is complementary to the template strand (3’ to 5’)

What is the difference between genome and gene?

genome: entire set of DNA

gene: segments of DNA that are transcribed into RNA, and then translated into protein

Some genes are expressed (copied into RNA) _______________ (all the time). Some genes are subject to _________________ (only copied into RNA under some conditions).

constitutively, regulated expression

What are the differences between messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA)?

mRNA: codes for the protein sequence

tRNA: non-coding (doesn’t code for a protein), components of the machinery that translate mRNA into protein

rRNA: a component of ribosomes, non-coding, facilitate translation of RNA into proteins

What are the 3 stages of transcription?

1. initiation: transcription is initiated at promoter sequences in the DNA template

2. elongation: RNA strand complementary to template DNA is synthesized by RNA polymerase

3. termination: RNA strand synthesis stops + RNA polymerase is released

_______________ direct the RNA polymerase to the proper site for transcription initiation.

Promoter sequences

What subunit of RNA polymerase helps it find the correct site for transcription?

sigma subunit

When is the sigma subunit released?

after transcription is initiated

RNA polymerase does NOT require a ___________.

primer

What is a distinct feature on the 5’ end of newly synthesized prokaryotic RNA?

triphosphate nucleotide

What does the transcription bubble contain?

RNA polymerase, unwound DNA, and newly forming RNA

For RNA transcription, in the elongation step, the DNA template strand is read in the ___________ direction and the newly synthesized RNA is made in the ___________ direction.

3’ to 5’, 5’ to 3’

What occurs during transcription termination in prokaryotes?

• formation of phosphodiester linkages stops

• RNA-DNA hybrid dissociates

• DNA re-anneals (binds)

• RNA polymerase releases

What are the two different ways that the formation of phosphodiester linkages stops?

1. intrinsic termination

   • in the RNA: formation of a ‘hairpin’ followed by a string of U’s

2. protein - dependent termination

What does E. coli use as as their source of carbon and energy? What do they use when this source is scarce in the environment?

glucose, lactose

What enzymes does E. coli express when lactose is present?

beta-galactosidase and permease

What is the purpose of lactose operon?

group of genes found in E. coli that enable the bacteria to use lactose as an energy source when its available

What are the Z and Y lactose operon genes?

genes that code for beta-galactosidase and permease respectively

What are the promoter, operator and i (gene) elements of the lactose operon?

P (promoter): where RNA polymerase binds to start transcription

O (operator): a DNA segment where a repressor protein can bind

i (gene): encodes a repressor protein that can bind to the operator and blocks RNA polymerase from moving

What occurs with the lactose operon when lactose IS NOT present?

• the i gene makes the repressor protein

• the repressor binds to the operator, blocking RNA polymerase from transcribing the structural genes

• RESULT: no enzymes for lactose digestion are made (no lactose to digest anyways)

What occurs with the lactose operon when lactose IS present?

• some lactose is converted into allolactose, which acts as an inducer

• allolactose binds to the repressor and, causing a structural change that reduces repressor’s affinity for binding to the operator sequence in the DNA

What is the lac operon an example of?

regulated expression in bacteria (Z and Y genes are only expressed in the presence of lactose)

How many RNA polymerases do eukaryotes have? What does each polymerase do?

3

RNA polymerase I: transcribes rRNA (ribosomal RNA)

RNA polymerase II: transcribes mRNA (messenger RNA)

RNA polymerase III: transcribes tRNA (transfer RNA) and some rRNA

What promoter elements are recognized by RNA pol II?

TATA box (consensus sequence)

initiator element (Inr)