Biochemistry I(CHE 418 / 5418

Reading Assignment

Berg et. al (2012) Chapter 4

β

Central Dogma of Molecular Biology

DNA RNA Protein mRNA. tRNA, rRNA, snRNA Nucleus

– Replication - DNA directed DNA synthesis

– Transcription - DNA directed RNA synthesis· Processing of mRNA capping, polyadenylation, splicing

• Cytoplasm– Translation - RNA directed Protein synthesis

Transcription Translation

Replication

Functions of Nucleic Acids

Building blocks of DNA and RNA– DNA = Genetic material– RNA = Adapter molecule between DNA and protein

Transport chemical energy within the cell– ATP

Signal molecule cyclic AMP

Nucleic Acids

• Nucleic Acid - linear, non-branched polymer of nucleotides

Classes of Nucleic Acids– RNA = ribonucleic acid– DNA = 2' deoxyribonucleic acid

Nucleotide• Nucleotide contains:

– Pentose sugar– Nitrogenous base– Phosphate

– One or more

Adenosine Triphosphate (ATP)

N

NN

N

NH2

O

OHOH

HH

HH

OPO

O-

O

PO

O-

O

P-O

O-

O

Nucleotide

• Pentose sugar– Carbons are numbered with primes to differentiate between

carbons / nitrogens of nitrogenous bases

β-D-2-DeoxyriboseDNA Only

RiboseRNA Only

OH

3' 2'

1'O

4'

OHOH

HHH

5'

H

HOOH

3' 2'

1'O

4'

HOH

HHH

5'

H

HO

Nucleotide

• phosphate group

O-P-O

O-

O

Pyrimidine Bases

Cytosine(C )

4-amino-2-oxypyrimidineDNA and RNA

Thymine(T)

5-methyl-2,4-dioxypyrimidineDNA ONLY

Uracil(U)

2,4-dioxypyrimidineRNA ONLY

Pyrimidine

N

NH

NH2

O

N3

2

N1

6

5

4

HN

NH

O

O

HN

NH

O

O

Purine Bases

Adenine (A)

6 – aminopurineDNA and RNA

Guanine(G)

2- amino-6-oxypurineDNA and RNA

N1

2

N3

4

56

NH

9

8

N7

N

N NH

N

NH2

HN

N NH

N

O

H2N

Purine

Sugar Phosphate Backbone

• Nucleotides connected by 3’ to 5’ phosphodiester bond– Imparts uniform negative charge to DNA / RNA

• Negative charge repels nucleophilic species (e.g. hydroxyl) thus the phosphodiester bond resists hydrolytic attack.

• Separation by agarose gel electrophoresis

– Creates 3’ and 5’ end (directionality) • Convention: Nucleotide sequences are written 5’ to 3,’ L to R

Bases are attached to sugar by Beta Glycosidic linkage

• N-9 of purine and N-1 of pyrimidine

Nucleotide

Nucleoside = sugar + nitrogenous base,

Nucleotide = sugar + nitrogenous base + phosphate.

N

NN

N

NH2

O

OHOH

HH

HH

OPO

O-

O

Adenosine monophosphate(A nucleotide)

Adenosine(A nucleoside)

N

NN

N

NH2

O

OHOH

HH

HH

OH

What data did Watson and Crick use to determine the structure of DNA

• X ray diffraction photograph of DNA crystals

• Chargaff’s rules

• Bond angles from reference books

• Built models

Erwin Chargaff’s “Rules”• Edwin Chargaff

determined the composition of DNA from many organisms– [A] = [T]

– [G] = [C]

DNA is a Helix• X ray diffraction

photograph– Maurice Wilkins

and Rosalind Franklin

– Two chains that wind in a regular helical structure.

Watson and Crick (Complementary) Base Pairing

A T

G C

Nucleotide content determinesmelting point of DNA.

Double Helix• B form

– Diameter of helix = 20.0 Å (2.00 nm)

– 10.4 base pairs / turn; 34 Å (3.4 nm)

– 1 base pair 3.4 Å (0.34 nm)

• Note– Complementary base pairing

– Major grove

– Minor grove

– Antiparallel

– Hydrogen bonding between complementary base pairs.

DNA held together by hydrogen bonding and Hydrophobic interactions

• Hydrogen bonding between base pairs– 4 – 21 kJ / mol (1 – 5 kcal/mol)

• Hydrophobic interactions (van der Walls) due to base stacking.– 2 – 4 kJ / mol (0.5 – 1.0 kcal / mol)

Forms of DNA• B form

– “Normal” form

– Watson and Crick form

• A form– “dehydrated” B form

– nucleotide tilted 20o relative to helical axis

• Z form (“zig zag”)– stretches of alternating purine /

pyrimidines

– base pairs flip 180o

– Left handed helix

DNA is Organized into Genes• Gene

– discrete, functional unit of DNA – when expressed, (transcribed) yields a

functional product • rRNA, tRNA, snRNA• mRNA - translated into a polypeptide sequence.

– Open reading frame - long stretch of nucleotides that can encode polypeptide due to absence of stop codons.

Chromatosomes Pack to FormChromatin Fibers

Histones H1, H2A, H2B, H3, H4

Histones contain (>20%) arg and lys ---basic amino acids

Karyotype

• Photograph of chromosomes from a single organism

• Arranged by size (largest to smallest)

• Homo sapiens – 46 chromosomes

– 23 pairs

• 3 billion base pairs (hapliod)

• 25,000 genes

Chromosome Contains• Centromere site that

connects sister chromatids

• Kinetochore attachment site of spindle to chromosome

• Telomere - nucleotide repeat at end of linear chromosome– TTAGGG x 1000

– synthesized by telomerase

Properties of DNA

• Melt / Anneal / Reanneal

• Hypochromic effects

• Supercoiled / Relaxed

dsDNA can Reversibly Melt• Heating DNA breaks

hydrogen bonding between base pairs.– Acid or base also works

• Tm = melting temperature– Half the helical structure is

lost

• Single stranded DNA absorbs light more efficiently than double stranded DNA

Hypochromic effect (Hypochromism)

• DNA can melt and then re-anneal.

• If sequences are similar, they will reanneal or hybridize.

DNA exist as Linear or Circular Molecules

• Prokaryotic, Mitochondrial and Chloroplast genomes are circular– Circular molecules may exist in

topological isomers • Relaxed

• Supercoiled

• Eukaryotic genomes are linear molecules

Single Stranded Nucleic Acids can form complex structures

• Stem Loops are produced by H-bonding between complementary regions in DNA and RNA.

• Mismatches are observed

• Hydrogen bonding stabilizes more complex structures.

• Often observed in ribosomal RNA molecules

Single Stranded Nucleic Acids can form complex structures

Replication

"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

Watson, J.D. and Crick, F.H.C., Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid, Nature, 171, pp. 737-738, (1953).

DNA may be labeled with 15N

• Grow E. coli in media containing 15NH4Cl and 14NH4Cl.

• Purify DNA• Separate DNA using density

gradient equilibrium sedimentation– CsCl gradient from 1.66 – 1.76 g

/ cm

Testing the Semiconservative Replication Hypothesis

• Matthew Meselson and Franklin Stahl (1958)– Grew E. coli in 15NH4Cl until DNA was completely labeled.

– Transferred E. coli to 14NH4Cl containing media.

– Followed labeling pattern of DNA through several generations using density gradient equilibrium sedimentation

DNA Replication• DNA directed DNA synthesis• DNA Polymerase

– adds deoxyribonucleotide units to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction.

• E. coli DNA Pol I isolated by Author Kornberg (1958)

– DNA Polymerase requires• Four dNTPs (dA,T,dG,dC)• Divalent cation (Mg2+)• Template DNA• Primer provides 3’ OH

DNA Polymerase Reaction Mechanism• Nucleophilic attack by the 3’ OH on the alpha phosphate group of

dNTP

• PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)

Types of RNA• Types of RNA

– Ribosomal RNA (rRNA)– part of the ribosome

– Transfer RNA (tRNA) -

– Messenger RNA (mRNA)– sequence translated into protein sequence.

– Small nuclear RNA (snRNA) – involved in splicing (spliceosome)

– Micro RNA (mi RNA) – small RNA complementary to mRNA that inhibits translation of the mRNA

– Small interfering RNA (siRNA) – small RNA that binds to mRNA causing destruction of mRNA

Transcription

• DNA directed RNA synthesis

• RNA Polymerase– adds ribonucleoside triphosphate units

to an existing DNA molecule in a template directed fashion in the 5’ to 3’ direction.

– RNA Polymerase requires:• Four NTPs (A,U,G,C)

• Divalent cation (Mg2+)

• Template DNA

• NO primer required

• Lacks endo and exo nuclease activity

• RNA molecules are complementary to the DNA template.

One prokaryotic RNA PolThree eukaryotic RNA Pol

RNA Pol IRNA Pol II

mRNARNA Pol III

Structure of RNA

• Genes may or may not be transcribed depending on the needs of particular cell type.

• gene is a functional region of DNA

– expressed genes are “TURNED ON”– unexpressed genes are “TURNED OFF”

RNA Polymerase Reaction Mechanism• Nucleophilic attack by the 3’ OH on the alpha phosphate group of

NTP (ribonucleoside triphosphates)

• PPi (pyrophophosphate) is hydrolyzed to Pi + Pi (orthophosphate)

RNA molecules are complementary to the DNA template.

• mRNA is complementary to template strand• mRNA is identical (except for U to T changes) to the

coding strand.

Prokaryotic Promotor

– Pribnow box (also called TATA box)• 5’TATAAT 3’ centered at -9/-10

– designated by the 5’ to 3’ sequence on the NONtemplate strand» 8 to 10 nucleotides left (5’ or upstream) of transcriptional start site

(designated +1 --- there is no 0 nucleotde)

– -35 sequence • 5’TTGACA3’ centered -35 from

Eukaryotic Promotor

• Class II genes– those synthesized by RNA Pol II.

• Pre mRNA and snRNA

• Parts– TATA or Hogness box– GC box (GGGCGG)– CAAT box

Transcriptional Termination• Rho dependent

– Involves protein called Rho

• Rho independent– Involves stem loop structure in

mRNA

– Stem loop is followed by UUUs

mRNA

• Prokaryotic mRNA are polycistronic– May encode two or more proteins

• Eukaryotic mRNA are monocistronic– Encode only one protein

Eukaryotic mRNA are Posttranscriptionally Modified

• Capping– attachment of 7-methylguansine using 5’ to 5’triphosphate linkage

• Polyadenylation– attachment of 40 to several hundred adenine nucleotides to 3’ end of mRNA

• Splicing– removal of introns

Amino acids are attached to 3’ end of tRNA

• Aminoacyl-tRNA synthestase– attaches amino acid to tRNA

Translation• Stages of Translation

– Initiation• assemble and align ribosome, mRNA,

and tRNAfMet

– Elongation• template directed synthesis of proteins

– Termination• termination factors halt protein

synthesis• ribosome, mRNA and new protein

dissociate

• Orientation of Translation– Ribosomes move 5’ to 3’

along mRNA– Protein is synthesized N to C

Genetic Code• Marshall Nirenberg, Har Gobind Khorana, Frances Crick

• Specific- Unambiguous– specific codon always codes for SAME amino acid– Three nucleotides (codon) = one amino acid

• 61 codons encode amino acids– Codons encoding one amino acid usually differ in the last base.

• 3 codons encode stop codons (UAA, UAG, UGA)

• Universal– conserved from species to species

• main exception = mitochondria

• Redundant (also called degenerate)– amino acid may have more than one codon

• Nonoverlapping and comma less (no puctuation)– read from fixed starting point (AUG)– lacks punctuation between codons

Codon Usage Table

• mRNA are “read” three nucleotides (codon) at a time starting from a fixed point.

Translational Start Site• AUG encodes Met (n-terminal amino acid).

– Prokaryotes use a Shine-Dalgarno sequence to align a ribosome on the mRNA upstream ( 5’) of AUG

– Eukaryotes use the 5’Cap to align the ribosome on the mRNA

Mitochondrial Genetic Code differs from the Universal Code

Eukaryotic mRNA contain Exons and Introns

Philip Sharp and Richard Roberts (1977)• Exons – coding regions• Introns –noncoding regions

– intervening sequences

Introns were discovered by hydridizing mRNA to genomic DNA

Splicing• Removal of introns• Spliceosome – specific proteins and small nuclear

RNA.• Most introns start with GU and end with AG

WHY UNDERSTAND TRANSLATION?• Many Antibiotics kill bacteria by inhibiting

prokaryotic translation!!!