Graduate Biochemistry

Topics:

Structure of Biological Molecules

Molecular interactions

Organization and Regulation of Molecular Complexes

Genes and their expression

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Chapter 1 - Origins of Life

Earliest signs of life ~3.5 billion years ago

The pre-life atmosphere contained H₂O, N₂, CO₂, CH₄, NH₃, and SO₂.

Lightning or ultraviolet radiation could have provided energy for reactions to form simple organic compounds.

An experimental test of this hypothesis showed that reactions of primitive atmospheric compounds could result in amino acids and other common metabolites found in modern day cells.

These compounds contain many of the functional groups necessary for modern day biochemical reactions.

Simple organic compounds - raw materials for a period of chemical evolution in which simple molecules condensed together to form larger functional units.

At some point, an RNA-like molecule may have arisen, which could direct its own replication.

Next major step: formation of a vesicle around the self-replicating molecules.

Enclosure of self-replicating systems within vesicles would form the first cells.

Allows the cell to maintain high local concentrations of necessary components,

which would otherwise diffuse away.

Early cells acquired ability to catalyze reactions for the synthesis of necessary compounds from simple precursors.

Acquired the ability to harvest energy for these reactions from their environment.

Ability of early cells to synthesize biological molecules, to utilize an energy source, and to direct their own replication, allowed them to propagate.

All organisms appear to be descended from a single common ancestor, based on conserved sequences of key cellular components, such as the ribosome.

Present-day organisms can be divided into three kingdoms of life:

Bacteria, Archaea, and Eukarya

Bacteria and Archaea:

Are commonly grouped together in the classification prokaryotes.

Unicellular and relatively simple internal architecture.

Enclosed in an outer cell membrane, but lack internal membrane compartments.

Internal cytoplasm appears to be organized into different regions, however.

Rendering of contents of E. coli cell. Note the organization of DNA and dense packing of cell contents, particularly ribosomes, the site of protein synthesis.

Typical cell: 70% H₂O

Dry weight mostly carbon – 98% of dry weight composed of C, N, O, H, Ca, P, K, and S.

Eukaryotes consist of the Eukarya

Distinct from prokaryotes in having a nucleus to encapsulate DNA.

Can be both unicellular and multicellular.

Eukaryotic cells characterized by organellar organization:

Nucleus – DNA

Endoplasmic reticulum – translation, translocation, modification

Golgi – post-translational modification

Mitochondria – aerobic metabolism (probably descended from bacterium)

Chloroplastc – plant photosynthesis (probably descended from bacterium)

Lysosomes and peroxisomes – degradative metabolism

Cytosol- the rest of the cytoplasm

Chapter 3: Nucleosides, Nucleotides and Nucleic Acids

Nitrogenous base – planar, aromatic, heterocyclic derivatives of purine or pyrimidine

Nucleosides are comprised of a nitrogenous base linked to a sugar

(usually ribose or deoxyribose)

Nucleotides are nucleosides attached to at least one phosphate group

Base Formula	Base (X= H)	Nucleoside (X= ribose)	Nucleotide (X= ribose phosphate)
purines
	Adenine Ade A	Adenosine Ado A	Adenylic acid Adenosine monophosphate AMP
	Guanine Gua G	Guanosine Guo G	Guanylic acid Guanosine monophosphate GMP
pyrimidines
	Cytosine Cyt C	Cytidine Cyd C	Cytidylic acid Cytidine monophosphate CMP
	Uracil Ura U	Uridine Urd U	Uridylic acid Uridine monophosphate UMP
	Thymine Thy T	Deoxythymidine dThd dT	Deoxythymidylic acid Deoxythymidine monophosphate dTMP

Nucleotide sugars are commonly ribose or deoxyribose

5’ Ribonucleotide – sugar=ribose	5’ Deoxyribonucleotide – sugar=2’-deoxyribose

Most nucleotides are found in polymers (RNA and DNA).

Free nucleotides often complexed with Mg⁺⁺ as a counterion

Some important nucleotides

H3PO4 +				+ H2O
	Adenosine diphosphate (ADP)		Adenosine triphosphate (ATP)

Adenosine triphosphate (ATP):

Most energy from cellular metabolism passes through ATP as an intermediate

Cellular concentration of ATP ~5 mM

But daily turnover of ATP is ≈ to total body weight

Energy of hydrolysis is used to drive other reactions.

Sometimes used to form intermediate necessary for further synthesis

Other important nucleotides and their uses:

Nicotinamide adenine dinucleotide - NAD	Flavin adenine dinucleotide - FAD

NAD⁺ and NADP⁺ can be reversibly reduced to form NADH or NADPH in oxidation-reduction reactions (nicotinamide is from vitamin niacin).

FAD also is used for oxidation-reduction (riboflavin portion is vitamin B₂)

All three act as electron transporters

Coenzyme A (CoA)- used as acyl group carrier. Pantothenic acid is vitamin B3.

Nucleic Acids

Phosphodiester bond links each nucleotide unit to form polynucleotide chain

Linkage between nucleotides is typically between 5’ O and 3’O

Terminii named 5’ and 3’ ends

Forms polyanion due to negatively charged PO₄

Is this RNA or DNA?

Double helix structure of DNA

DNA base composition:

Chargaff’s rule: For any organism %A=%T and %G=%C.

But %G+C can vary widely (25 – 75%)

Watson and Crick used model building to describe structure of DNA

Explained Chargaff’s rules

Fit fiber diffraction measurements of DNA

3-D structure of DNA	2-D representation showing H-bonds
DNA Kinemage 1: B-DNA	DNA Kinemage 2: Base pairs.

Features of the double helix:

Two polynucleotide chains wrapped around a common axis -> double helix

Strands are antiparallel but both are right-handed

The bases are buried in the core of the helix, sugar-phosphate backbone is exposed to solvent

Interior bases partially accessible through major and minor grooves of double helix

Bases form characteristic sets of hydrogen bonds (sharing of proton by dipoles) between complementary bases:

A pairs with T, G pairs with C

termed complementary base pairs

Accounts for Chargaff’s rules

	Genome sizes:
	Eukarya
	Homo sapiens	3,127,936,000
	Arabidopsis thaliana	117,950,579
	Caenorhabditis elegans	100,237,364
	Schizosaccharomyces pombe	12,515,113
	Saccharomyces cerevisiae	12,162,624
	Bacteria
	Escherichia coli K12	4,639,221
	Haemophilus influenzae Rd	1,830,138
	Mycoplasma genitalium	580,074
	Archaea
	Methanococcus jannaschii	1,664,970
	Viruses
	Human immunodeficiency virus 1	9181
	Simian virus 40	5243
	Bacteriophage l	48502
	Bacteriophage T4	168,903
	Bacteriophage T7	39937

Suggested mechanism for replication of DNA based on complementarity of bases:

One strand can serve as template for synthesis of new copy of its complement

DNA polymers can be enormous.

Total DNA of cell is its genome

Genomes may be divided among several molecules (chromosomes)

Most prokaryotes have only one double strand copy of their genomes per cell – they are haploid

Most eukaryotes have not only haploid cells,

but also diploid cells that have 2 copies of each double-strand chromosome

The structure of double-strand DNA is relatively unaffected by the DNA sequence

Single stranded DNA and RNA tend to try to form basepairs with other regions of the molecule that are complementary, so structure can vary for different sequences.

Nucleic acid function

Avery, MacLeod and McCarty

Isolated DNA from pathogenic strain of D. pneumoniae,

mixed it with non-pathogenic strain

recipient strain was transformed (permanently changed) to pathogenic

ð DNA is the source of genetic information

How is the genetic information in the DNA expressed by the cell to affect the way it behaves?

DNA directs transcription of messenger RNAs (mRNAs)

mRNA is translated into protein at the ribosome by base-paring to aminoacyl-tRNAs

The Central Dogma: DNA->RNA->protein	Translation of mRNA into protein

So if the DNA sequence is altered by mutation

change can affect the mRNA sequence

which can in turn change the protein sequence.

Proteins perform much of the chemical and structural functions of the cell, either directly or indirectly.

Most enzymes (biological catalysts) within the cell are proteins, but a few are made of RNA.

Brief intro to Recombinant DNA (DNA cloning)

Key to cloning - the restriction-modification system of bacteria

Most bacteria can bind and absorb DNA from the environment - transformation

Probably an adaptation to allow individuals to acquire variant genes from others of their species.

But if DNA from another species, could wreak havoc with regulation of gene expression.

Bacteria have a biochemical system that allows them to acquire DNA of their own species, but not from others- The restriction-modification system.

Each species (or strain) of bacteria has a distinct pair of enzymes:

A site-specific DNA methylase (adds a CH₃- group to a particular type of nucleotide)

and an endonuclease, which cuts DNA that is not methylated at that site.

(Endonuclease – cuts within DNA [or RNA] strand

Exonuclease – removes nucleotides from ends)

Restriction endonucleases have been purified from 100s of different species

Most recognize palindromic sequences (sequences that looks the same on either strand from either direction)

E.g.

Bacterial strain	Endo.	Site
E. coli R	EcoR I	5' GAATTC 3' 3' CTTAAG 5'	cuts 3' of G leaves 5' overhang
H. influenzae	HindIII	5' AAGCTT 3' 3' TTCGAA 5'	cuts 3' of 1st A leaves 5' overhang
H. aegyptus	HaeII	5' RGCGCY 3' 3' YCGCGR 5'	cuts 3' of last C leaves 3' overhang

Used in many ways in DNA cloning, especially to cut vector DNA and gene of interest for splicing gene into vector:

"sticky ends" from EcoRI cut of one fragment can base pair with those from another

isolate fragment with insert from gel, anneal sticky ends to plasmid DNA, and ligate with DNA ligase.

DNA ligase - DNA repair enzyme

repairs breaks in DNA backbone

Requires high energy cofactor.

Usually use enzyme from T4 phage, which can ligate blunt or sticky ends (uses ATP).

E. coli enzyme requires NAD+ instead of ATP and only repairs sticky ends.

Plasmids

autonomously replicating circular minichromosomes

most derived from drug-resistant bacteria

have origin of replication for amplifying DNA and marker gene to select for cells carrying it

Automated DNA sequencing:

Once cloned, DNA can be sequenced by either chemical or enzymatic methods. Chemical method is still used for footprinting type experiments, but virtually all sequencing is performed using DNA polymerase and chain terminating nucleotides.

Enzymatic sequencing reaction

Anneal (hybridize) single strand ssDNA to a specific oligonucleotide primer (complementary to flanking vector sequence, or to known sequence within the insert DNA). The oligonucleotide serves as primer for synthesis of DNA by DNA polymerase, using the plasmid as template.

Usually an unlabelled oligonucleotide is used, plus all 4 deoxynucleotide triphosphates (dNTPS)

Plus a mixture of different 2'-3'-dideoxynucleotide triphosphate (ddNTPs), each of which has a characteristic fluorescent label attached.

ddNTPs can be incorporated by the polymerase, but cause synthesis of the chain to stop,

since they have no free 3'-OH to serve as acceptor of 5' PO₄ in the polymerization reaction.

Thus, when a ddNTP is incorporated into the DNA product, the DNA chain cannot be extended and a specific colored label is incorporated at the same time.

DNA products are denatured (separated from the template) and the products are separated according to size by electrophoresis on a polyacrylamide gel or capillary tube.

As the samples run off the bottom of the sequencing gel, they are detected by the laser fluorimetry and automatically recorded.

polymerase: usually thermostable polymerases and “cycle sequencing” similar to PCR.

Automated sequencing of genomes of humans and other model organisms is revolutionizing the way molecular biology is done. The other player in this revolution is the Polymerase Chain Reaction

Polymerase Chain Reaction (PCR)

PCR is the amplification of a specific DNA fragment in vitro, directed by using a pair of oligonucleotides to prime DNA synthesis of the region between the primers using DNA polymerase.

Successive rounds of DNA synthesis are accomplished by first heating the template DNA and primers to 94° to denature the template (separate strands),

cooling to approximately 50° to allow the primers to anneal (base pair) to the template,

followed by a polymerization reaction using DNA polymerase

The cycle of DNA denaturation, annealing and synthesis and polymerization is repeated many times.

Each time the reaction is repeated, the products of the previous cycle are used as template, as well as the original template, causing an exponential synthesis of the fragment of DNA between the primers.

This process was greatly facilitated by the use of thermostable DNA polymerases and automatic temperature cylers.

Using this procedure, a single DNA molecule can be specially amplified and detected.

Used in a myriad of ways in molecular biology.

Need not know sequence of DNA between the primers,

so can amplify DNA of related organism based on primers of regions known to be conserved.

Or use sequence identified from genome sequencing projects

Sensitive method to detect small amounts of contaminating bacteria, mutants cells, etc.

Can be used in combination with reverse transcriptase to amplify specific mRNA sequences.

Choice of polymerase is again important. Thermus aquaticus (Taq) most commonly used, but it is error-prone, lacks proofreading 3'->5' exonuclease and loses activity after about 35 cycles at high temperature.

Other organisms, like Pyrococcus furiousus, produce polymerases that are more thermostable and less error prone and have proofreading function. Allowing more faithful replication of longer genes (up to 40 kb)

Site directed mutagenesis

Many different strategies, all require: synthetic oligonucleotide(s) with desired mutation extending oligonucleotide by DNA polymerase to reconstruct entire sequence Animated version of figure

strategy for eliminating starting “wild-type” template

e.g. “quick change” strategy:

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