biochem 10 Nucleic acids

when was DNA discovered and by who

1869

friedrich Miescher

what was DNA first called

nuclein (since found in nucleus of white blood cells)

what does it mean for nulcleic to be a polymeric chain

made of many monomers linked together

what are the monomers of a nucleotide

sugar (ribose or deoxyribose), nitrogenous bases, and a phosphate group

monomer units are connected by ______________ bonds (specific link:___________-)

covalent; phosphodiester link

how do we read a nucleic acid chain

5’-3’

nucleotide functions (3)

• energy for metabolism (ATP)

• enzyme cofactors (NAD+)

• signal transduction (cAMP)

nulceic acid functions

• storage of genetic information (DNA)

• transmission of genetic info (mRNA)

• processing of genetic info (ribozomes)

• protein synthesis (tRNA and rRNA)

a nucleotide contains

nitrogenous base. sugar, phosphate group

nucleoside contains

nitrogenous base and sugar

nucleobase contains

nitrogenous base

nitrogenous bases are either

pyrimidines or purines

pyrimidines (which ones are these; structure)

• cytosine

• uracil

• thymine

planar, single hexagone

purines (structure, which ones are these)

• adenine

• guanine

a hexagone+pentagon

almost planar

uv light absorption of nitrogenous bases

250-270 nm

UV light absorption is used to detect and quantitate nucleic acids

what are the pentose sugars in nucleotides

either a ribose or a deoxyribose

what identifies if something is DNA or RNA

if it has ribose or deoxyribose

EVEN IF deoxyribose has uracil (STILL DNA)

ribose vs deoxyribose

ribose has a oxygen on the 2C’ and is in RNA

deoxyribose has no oxygen on C2’ and is in DNA

how does the sugar bind to the nitrogenous base

if it is a purine, C1’ binds to N9

if it is a pyrimidine, C1’ binds to N1

it is a COVALENT —> beta-N-glycosidic bond

adenosine functions as a what (3) and what does it cause (4)

• functions as a autocoid/ local hormone, and neuromodulator in which it is in the bloodstream influencing blood vessel dilation, smooth muscle contraction, neurotransmitter release, and fat metabolism

• is also a sleep regulator where rises when we wake up to promote an eventual sleep (caffeine blocks it)

nucleotides are (another name)

nucleoside phosphates

most nucleotides have ribose or deoxyribose?

ribose —> ribonucleotides

phosphate group (charge)

negative charged at neutral pH

phosphate group attached to sugar how

attached in the 5’ position

how are nucleic acids built (where energy)

5’ triphosphates like ATP, GTP, TTP, CTP

atp does what

central to energy metabolism

GTP does what

drives protein synthesis

CTP does what

drives lipid synthesis

UTP does what

drives carb metabolism

nucleic acids contain ___ phosphate moiety (part) per nucleotide

one

how are nucleic acids formed (the bonds)

covalent bonds formed by phosphodiester linkages

the backbone of a nucleic acid is _______ charged

neg charged (at pH 7)

the backbone of nucleic acid consist of

the phosphate group and the pentose sugar

DNA Backbone (stability and hydrolysis)

fairly stable

subject to slow hydrolysis (when no enzyme present)

accelerated hydrolyses by enzyme DNAse

The hydrogen instead of OH in RNA is more stable (not broken apart as easily)

nucleic acids structure in terms of branching

linear and no branching

RNA backbone (stability, hydrolysis)

stable but rapidly hydrolyzed in alkaline conditions (DNA IS NOT)

-in water, RNA last a few years but in a cell it mRNA is degraded in a few hours due to OH group on C2’

nucleic acids directionality (differences on ends)

5’-3’

the 5’ end lacks a nucleotide (ends with the sugar attached to is phosphate)

the 3’ lacks another phosphate on the C3’ of the pentose sugar (lacks a nucleoside)

hydrolysis of RNA

unstable in alkaline conditions

can by hydrolyzed by enzyme RNase

what is dicer

is an enzyme that cleaves double-stranded RNA (dsRNA) into oligonucleotides

-protection from viral genomes

RNase is a what, how common, example of one

• RNase enzymes are abundant around us:

  •  S-RNase in plants prevents inbreeding

  • Dicer is an enzyme that cleaves double-stranded RNA (dsRNA) into oligonucleotides

    • protection from viral genomes

MECHANISNS OF BASE CATALYZED RNA HYDROLYSIS

The -OH will want to pull the H from OH on the sugar to make water. So then the other O doesn’t have partner, and will attack the phosphate. Then phosphate breaks bond with the oxygen that attaches to the lower sugar, and then the 2 nucleotides that were together have broken into 2

how was DNA discovered (2)

• One of the most important discoveries in biology

• DNA extracted from a virulent bacterium was injected into a nonvirulent strain of the same bacterium

  • The nonvirulent strain became virulent (1940s)

• Chargaff found that the 4 nucleotide bases of DNA occur in different ratios in the DNAs of different organisms and that the amounts of certain bases are closely related (late 1940s)

  •  Clue to how genetic information is based down

• Franklin and Wilkins used X-ray diffraction to analyze DNA (early 1950s)

how did we figure structure of DNA

• Franklin and Wilkins used X-ray diffraction to analyze DNA (early 1950s)

chargraff’s rules of DNA (4)

1.  The base composition  (A,G,C,T) of DNA generally varies from one species to another

2.  DNA specimens isolated from different tissues of the same species have the same base composition.

   1. So sample of skin and then my adipose tissue, they have the same base composition because same species/ person

3. The base composition of DNA in a given species does not change with an organism’s age, nutritional state, or changing environment.

   1. Genetic code itself does not change. Genes off or on can, but not the code

4. In all cellular DNAs,

   1.  the number of adenine residues is equal to the number of thymine residues (A=T)

   2.  the number of guanine residues is equal to the number of cytosine residues (G=C).

   3.  So, the sum of the purine residues and pyrimidine residues is: A+G = T+ C

what is the equation for bases

A+G = T+C

SUM OF PURINES = SUM OF PYRIMIDINES

-because A pairs with T and G pairs with C

so if A is 30 then T is 30 and then that means that is 60. So C would be 20 and G 20. So 20 + 30 is 50 (A+G) and 20+30 is 50 (T+C)

how do bases bond with each other in DNA

hydrogen bonds

what bonds with what, how many bonds

PURINES PAIR WITH PYRIMIDINES

A with T —> 2 bonds

C with G —> 3 bonds

A with U —> 2 bonds (mRNA)

who was rosalind Franklin, what did she do

• Best known for her work on the X-ray diffraction images of DNA which led to the discovery of the DNA double helix

watson and crick model of DNA

• Missing layer means alternating pattern (major 22A & minor groove 12A)

• Hydrogen bonding:

  • A pairs with T

  •  G pairs with C

•  Double helix fits the data!

• Watson, Crick, and Wilkins shared 1962 Nobel

complementary strands of DNA (4 things- characteristics and bonds)

• the Two chains differ in sequence

  • Sequence is read from 5’ to 3’

• the Two chains are complementary

  • When they come together = hybridize

• the Two chains run antiparallel

  • i.e. one chain is 5’ to 3’ the other 3’ to 5’

• the two chains Held together by 2 forces:

  • Hydrogen bonding between bases

  •  Base-stacking interactions

what are the 2 interactions that hold the 2 chains together AND how to they make it stable

hydrogen bonds and base staking bases (dipole-dipole and vander waals interactions)

Help minimize contact of bases with water. The bases are more hydrophobic so want to be inside

DNA replication

-strands separate by helicase

-each strand serves as a template for the new strand

-DNA polymerase catalyzes the synthesis of new strands (in the 5’-3’ direction)

-lagging strands still goes in 5-3’ direction but is build in Okazaki fragments going 3’-’5 direction

-ends up with 2 DNA molecules, each one with a parent and a daughter strand

steps of DNA replication (wether it is DNA or RNA)

initiation, elongation, termination

DNA IN CELLS OCCURS IN THE FORM OF

chromosomes

DNA--> Histones --> chromosomes

how is information transferred in cells

-transcription and translation are

transcription

information encoded by the DNA molecule is transcribed via synthesis of mRNA

translation

3 base codons on the mRNA corrsponding to the specific amino acids directs the sequence of building a protein. tRNA brings them to the ribosome to form the amino acid chain.

what is the central dogma

A theory that states that genetic info flows only In one direction: from DNA--> RNA--> protein OR from RNA --> directly to protein

So replication--> transcription--> translation

DNA vs RNA (in types)

DNA- one type , one purpose

RNA - several types and purposes

• rRNA

• mRNA

• tRNA

• small nuclear RNA (snRNA)

• small RNAs (RNAi, miRNA, snoRNA, sRNA)

• long nocoding RNA (lincRNA and lncRNA)

ribosomal RNA function

 the basis of structure and function of ribosomes

messenger RNA frunction

carries the message for protein synthesis

transfer RNA function

carries the amino acids for protein synthesis

messenger RNA (made how)

using DNA template

mRNA (components)

-ribose instead of deoxyribose

-uracil instead of thymine

one mRNA may code for how many proteins

more than one

how does mRNA length vary depending on how many genes its coding for

• AND concentration (amount) correlates with

•  mRNA from a specific gene is usually always the same length

•  mRNA from different genes varies in length

•  mRNA cytoplasmic concentrations (how much) correlate with protein synthesis

tRNA works how

 tRNA are covalently linked to an AA and join with the mRNA to add the AA to the growing polypeptide (location: ribosomes)

MONOSCISTRONIC VS POLYCISTRONIC MRNA

mono- one gene

poly- codes for multiple genes

• Minimum length of mRNA is set by the length of the polypeptide chain for which it codes

  • Ex: 100 AA polypeptide requires 300 nucleotide mRNA coding sequence

in eukaryotes, DNA is transcribed to produce

 heterogeneous nuclear RNA (hnRNA)- pre mRNA

•  mixed introns and exons with poly A

•  intron = intervening sequence

•  exon = coding sequence

•  poly A tail - stability? Added on to the end and we think it has to be with stability while removing introns

introns and extrons

introns are those we get rid of

extrons are those we keep

what is splicing

Splicing produces final mRNA without intron

what does rRNA provide

 PROVIDES THE STRUCTURE AND FUNCTIONAL FOUNDATION FOR RIBOSOMES

ribosomes structure and components

-Ribosomes are about 2/3 RNA, 1/3 protein

- rRNA serves as a scaffold for ribosomal proteins

role of ribosomes

•  biosynthesis of proteins

  •  Genetic information in the nucleotide sequence of mRNA is translated into the amino acid sequence of a polypeptide chain by ribosomes

ribosomes in prokaryotes vs eukaryotes

tRNA is composed of what

• Small polynucleotide chains 73 to 94 residues each

  • Several bases usually methylated

• Each AA has at least one unique tRNA which carries the AA to the ribosome

translation (explain the process)

the genetic code (and start and stop codons)

• Different codons, or base triplets, correspond to each amino acid residue

  •  Wobble effect

• The AUG start codon places a Met at the beginning of eukaryotic proteins

  •  Often removed by proteases

• Three codons, UAA, UGA and UAG, are stop codons, which signal termination of translation

what is the wobble effect

Allows for some wiggle room with mutations since like AGU and AGC both code for Serine

what does non-coding RNA refer to

 commonly employed for RNA that does not encode a protein

what RNA’s are noncoding RNA’s

RNAi, small interfering RNA, microRNA

RNA interference (RNAi)

 Attracted considerable attention because their role in gene regulation makes them likely targets for drug discovery and development

which is blocking the synthesis of RNA

siRNA

• Exogenous (not from the body that somehow gets into the cells)

  double-stranded RNA that is taken up by cells

•  Typically binds perfectly to its mRNA target in animals

miRNA

• Single-stranded molecule from endogenous (we make in our bodies) noncoding RNA

•  Can inhibit the translation of many different mRNA sequences because its pairing is imperfect

• Imperfect match to mRNA which allows to inhibit multiple protein synthesis

miRNAs in glucose homeostatis

do nutritional components change your DNA?

NO, they impact the way DNA is expressed--> gene expression

•  Hormones are influenced by dietary factors

• Clear relationship between excessive CHO (mostly simple) consumption and lipid biosynthesis

2 ways in which hormones can impact gene expression and protein synthesis

direct method (fat soluble) and cell surface receptors (water soluble)

fat soluble hormone action on gene regulation

Direct method: the thing can go into cell and nucleus and bind to receptor on DNA

• Fat soluble go into cell

polar hormone action on gene expression

• Cell surface receptors : attach to receptor--> sends cascades--> lead to turning on or off of gene 

  • Water soluble target cell receptor

what vitamin A molecule goes directly into nucleus to affect gene regulation

retinoic acid

what is denaturation

is talking about unfolding and breaking noncovalent bonds

DNA denaturation is promoted by

• Electrostatic repulsion (phosphate group negative charge) between chains (charge neutralized to some extent by anions: Na+, K+, Mg2+);

  • High ionic strength stabilizes duplex structure

  • Using high salt to separate the strands

    • Backbone is negative, so if we add a lot of negatives, they will repulse as neg doesn’t want neg.

    • Or positive and positive

•  Altering pH or temperature (increase)

when DNA denatures, what remains intact

covalent bonds (all but the hydrogen bonds holding complementary bases together)

-so the phosphodiester bonds between sugar and phosphate, and the glycosidic bond between the sugar and the base

Genetic code remains intact

in DNA denaturation, what is borken

• Hydrogen bonds are broken

  • Two strands separate (denature)

  •  Base stacking is lost (the ladder steps)

  • UV absorbance increases = hyperchromic affect

what is the hyperchromic affect

because the bases in single-stranded DNA are more exposed (due to denaturation), they can absorb more light than the same bases in double-stranded DNA.

is denaturing reversible

Denaturation may be reversible: annealing (aka renaturation

what is annealing

reversible denaturation

the rate of DNA annealing depends on (4)

• Temperature

• Length and concentration of DNA

• Salt concentration

• Properties of sequence (C -G and A -T)

DNA is denatured at what temps (not PCR)

specific Temps (melting points)

Each DNA molecule is unique.

when temp is normal DNA is a __________, when does it denature, when does it anneal?(what temps- not specific numbers)

aka melting

• DNA exists as double helix at normal temperatures

•  Two DNA strands dissociate at elevated temperatures

• the strands reanneal when temp is lowered