Unit 2: Nucleic Acids

the closest living relative to humans is

chimpanzees

we share 99.5% of our genome with them

epigenetics involves

covalent modifications to DNA to alter gene expression

DNA function

to store genetic information

• gets transcribed by forming mRNA

• replication translation, transcription

there are many types of

RNAs

• miRNA, tRNA, rRNA, mRNA, etc

tRNAs have ____ attached to them

AAs, so can be charged

nucleobases in RNA

cytosine

guanine

adenine

uracil

C,A,G,U

nucelobases in DNA

cytosine

guanine

adenine

thymine

C,G,A,T

pyrimidines

• have only one ring

• includes C, U, T

purines

• have two rings

• includes A, G

RECOGNIZE structures of all nucelobases

nitrogenous bases

nucelobases

CUTAG

nitrogenous bases absorb

nitrogen-containing heterocyclic aromatic amines

• are mostly planar due to much sp2

• absorb UV light around 250-270nm due to the resonance in their aromatic rings

• so they are good H-bond acceptors and donors

• nitrogenous bases maximally absorb UV light at 260nm

how nitrogenous bases absorb UV

have purity ratios between 1.8-2 if pure

if there is protein contaminating the sample, the purity ratio would be greater than 2

• look at the absorbance when wavelength equals 260 and 280. If values of absorbance (260 absorbance/280 absorbance) is 1.8-2, it shows purity

nucleobases

aka nitrogenous bases

nucleosides

contain

• the nitrogenous base

• pentose sugar

nucleotides

contain

• nitrogenous base

• pentose sugar

• phosphate (at least 1)

nucleotide structure

• phosphate is off the 5’ carbon of the pentose sugar

• the alpha carbon is in the nitrogenous base

• the 1’ carbon is in the pentose sugar

ATP full name

adenosine triphosphate

Nucleoside name of adenine

adenosine deoxyadenosine

nucleoside name of guanine

guanosine deoxyguanosine

nucleoside name of cytosine

cytidine deoxycytidine

nucleoside name of thymine

thymidine OR deoxythymidine

nucleoside name of uracil

uridine

ribose

has a 2’ OH

ribonucleic acids

polymers of ribose

deoxyribose

has a 2’ H

deoxyribonucleic acids

polymers of deoxyribose

nucelic acid functions

• store genetic info in DNA

• transmit genetic info using RNA

• synthesize proteins (using tRNA and rRNA)

• process pre-mRNA (using small nuclear RNA)

parts of ATP

ATP has 3 Ps

ADP has 2 Ps

AMP has 1 P

adenosine has no Ps

nucleotide functions

• energy for metabolism (ATP)

• enzyme coenzymes (like NAD+)

• signal transduction (cAMP)

cAMP is made from

ATP

• it is ATP with two less ps (so only 1 P)

NAD+ is

two nucleotides joined together

glycosidic bond

between a sugar and nitrogenous base of a nucleotide

• is sterically hindered in nitrogenous bases

• can be syn or anti conformation

what conformation of glycosidic bonds is favorable in normal DNA?

anti

• helps form the DNA double helix

what conformation of glycosidic bonds is favorable in RNA

both syn and anti since RNA is single stranded and therefore more flexible

nucleic acid backbone uses what connections

phosphodiester bonds

• between the 3’ OH of a deoxyribose and the 5’ of the phosphate group of another nitrogenous base

• new nucleotides are added onto the 3’ hydroxyl end

• two phosphates leave to drive the condensation of two nucleotides (so a water molecule is released as a result)

the nucleic acid backbone is _____ charged

negatively

• due to the neg phosphates

the nucleic acid backbone is read

5’—>3’ (N—>C)

and made 3’—>5’. (C—>N)

RNA folding

secondary folding like hairpin loops all go together to form the finally completed tertiary structure

• the secondary structures are held together by hydrogen-bonds between A and U, G and C, and sometimes G and U

  • so between complementary bases

tertiary structure of RNA

see fully folded up in bottom right

primary structure of RNA

the single strand

hydrogen bonds between _____ form the secondary structures in nucleic acids

A and U

G and C

sometimes G and U

what can cause RNA to undergo hydrolysis/breaking apart?

alkaline (basic conditions)

enzymes (RNases)

• both cause the acidic H of the 2’ OH to be removed, so it attacks the adjacent phosphate group 

• this causes the phospodiester bond to break, so the two nucleotides split

• DNA is less susceptible to hydrolysis

strands in DNA run

antiparallel

where are phosphate groups in regards to the dsDNA

they are in the exterior, pointing out

nucleobases in the middle of dsDNA are ____ to the helical axis

perpendicular

interactions within dsDNA

hydrogen bonds between C and G (3 h-bonds) and A and T (2 h-bonds)

VDWs forces between the hydrophobic nucleobases

the number of adenosine residues in dsDNA equals

the number of thymidine residues

• and number C equals number G

• so number purines=number pyrimidines

the double helix of DNA is a _____ structure

secondary

• and the final structure of DNA (that we will cover)

base pairs within DNA ___

hydrogen-bond to their complementary counterpart

see where between two nucleotides h-bonds actually occur

major and minor grooves and what binds

major: base pairs are more exposed, so proteins that regulate gene expression bind here

minor: hard for things to reach the bases inside the dsDNA, so proteins that help package up DNA bind here

the major groove of DNA is large enough for _____ to bind

an alpha helix of a protein

• so regulatory proteins/transciption factors can bind here to recognize the pattern of nucleobases

how tightly DNA is bound in chromatin, for exmaple, regulates

gene expression

3 types of DNA strands

A form

B form (standard)

Z form

A form of DNA

• RHH

• has a larger opening in the middle

• occurs when you put DNA in a solution than let it dry

• has nucleobases tilted a bit, but still has major and minor grooves

B form of DNA

• RHH

• standard form

• bases are perpendicular to the helical axis

• has normal major/minor grooves

Z form of DNA

• LHH

• zigzagged helix

• bases are mostly perpendicular to the helical axis

what form of DNA is the major conformation

B

denaturation of DNA

causes dsDNA to turn ss

• can be caused by temp, pH, or ionic strength

• incr ionic strength (salt conc) or G/C content (since they have 3 not 2 h-bonds) causes the melting temp of the DNA to incr

its easier to anneal ____ regions of DNA

shorter

• since less room for error

what can cause DNA to anneal back to dsDNA?

• noraml temp/pH

• annealing takes a while

denaturation of DNA is

quick

can be caused by

• high temp

• low salt conc

• low pH

hyperchromic effect

a decr in the absorbance at 260nm (A260)

• seen when DNA/RNA is denatured since free nucleotides absorb more than ds or ss dNA

• since stacked bps in dsDNA absorb less light since they are covered up more

when DNA is heated, its UV absorbance…

incr

• since denaturation causes there to be more free nucleotides, and therefore absorbs more UV light (by 30-405)

Tm

the temp at which half of nucleotides are in dsDNA and half are in ssDNA

if temp lowers, absorbance ___

drops

• shows that dsDNA formed again after denaturation

melting curve of DNA

shows that there is most in ssDNA at high temp (and it has the highest UV absorbance here)

longer DNA has ___ Tm

higher

• since there is a higher temp at which half of its in ssDNA

Tm depends on

pH and ionic strength, and base composition

high salt conc: causes hydrophobic interactions within the dsDNA to solidify/incr (so Tm incr)

pH: too high or low pH affects Tm

base pair composition: high C-G bonds (triple h-bonds) causes Tm to incr

• since it takes more energy to break