Graduate Biochemistry

Topics:

Structure of Biological Molecules

Molecular interactions

Metabolism of Biomolecules

Energy utilization

Organization and Regulation of Molecular Complexes

Genes and their expression

 

Graduate Biochemistry Homepage

Chapter 1 - Origins of Life

Earliest signs of life ~3.5 billion years ago

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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.

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

 

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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.

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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.

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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 Ð 98% of dry weight composed of C, N, O, H, Ca, P, K, and S.

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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 Ð DNA

Endoplasmic reticulum Ð translation, translocation, modification

Golgi Ð post-translational modification

Mitochondria Ð aerobic metabolism (probably descended from bacterium)

Chloroplastc Ð plant photosynthesis (probably descended from bacterium)

Lysosomes and peroxisomes Ð degradative metabolism

Cytosol- the rest of the cytoplasm


 

Chapter 3: Nucleosides, Nucleotides and Nucleic Acids

 

Nitrogenous base Ð 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

5Õ Ribonucleotide Ð sugar=ribose

5Õ Deoxyribonucleotide Ð sugar=2Õ-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 Å 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:

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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

 

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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 5Õ O and 3ÕO

Terminii named 5Õ and 3Õ ends

Forms polyanion due to negatively charged PO4

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Is this RNA or DNA?

 

Double helix structure of DNA

DNA base composition:

ChargaffÕs rule: For any organism %A=%T and %G=%C.

But %G+C can vary widely (25 Ð 75%)

 

Watson and Crick used model building to describe structure of DNA

Explained ChargaffÕs rules

Fit fiber diffraction measurements of DNA

 

 

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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 ChargaffÕs rules

 


 

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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 Ð 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

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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.

 

Recombinant DNA (DNA cloning)

 

Means of amplifying DNA sequence of interest.

Use to determine DNA sequence

Overproduce gene product

Manipulate gene product (e.g. make mutations for study)

Make reagents for further study (determine fate of gene product in cell or analyze it in other individuals i.e. forensics)

 

 

Need some kind of vector to amplify your DNA:

In vivo:

Plasmids (typically)

Virus (bacteriophage, eukaryotic)

Hybrids

Artificial chromosomes (YACs)

In vitro:

PCR primers

 

Usually trying to clone into E. coli plasmid vector

 

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.

 

So, 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 Ð cuts within DNA [or RNA] strand

Exonuclease Ð removes nucleotides from ends)

 

So if you transform E. coli strain R with DNA from strain R

Select for expression of an acquired  gene --> colonies

But if you use DNA from strain K --> no colonies

 

The DNA from stain R is methylated by the EcoR I methylase

this keeps the EcoRI endonuclease from cutting the DNA

the DNA from strain K is methylated at different sites

so EcoRI can cut --> less efficient transformation

 

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

 

Modern plasmids have variety of features to make life easier.

Usually Ampicillin resistance gene, beta lactamase, as selectable marker (only cells with plasmid can grow).

Polylinker sequence - synthetic sequence with many restriction endonuclease cut sites,

Beta galactosidase gene fragment

Plasmids without inserts produce beta-gal and appear blue on X-gal plates.

Insert DNA disrupts open reading frame -> no beta gal -> white colonies

Plasmids introduced into cell by transformation:

Cells permeablized by osmotic shock or electrochemical pulse.

Circular DNA transforms efficiently

linear does not (due to cellular exonucleases)

Larger DNAs not as efficiently taken up as smaller ones for chemically permeablized cells.

Host cell usually defective for restriction modification system, so exogenous DNA not digested.

Other vectors:

Bacteriophage:

Lambda phage has 48.5 kb genome, packages one headful per virus particle.

Can remove middle third of genome to allow insertion of up to 16 kb of insert

Convenient for construction of Genomic and cDNA libraries


 

 


Cosmids:

Hybrid of lambda and plasmid, contains only packaging signal from lambda plus usual plasmid replication and marker genes.

Can insert up to 45 kb DNA

Use lambda packaging system to efficiently introduce large plasmid into cell.

Replicates as plasmid thereafter.

 

P1 phage: allows replication of up to 150 kb of DNA. Great for genomic libraries.

 

Expression vectors

Include promoter sequences to allow expression of mRNA and protein from insert DNA. Often are expressed as fusion proteins, your gene is fused with coding sequence for another protein. Can make more stable protein than foreign gene by itself. Also provides a handle for purification

ex.

Fusion product                     affinity resin

glutathione-S-transferase         glutathione

polyHis                                   Ni++

 

For bacterial expression, common promoter systems are lac and T7 phage

 

But many eukaryotic proteins not properly expressed and modified (e. g. glycosylation) in bacteria, so eukaryotic expression systems are used. Common promoters are SV40 and CMV in mammalian cells.

 


Reporter plasmids

 

Clone control region of your gene (promoter) upstream of gene encoding enzyme that is easy to monitor.

Can look at effects of changes in cell environment on expression of reporter gene to infer effects on the intact gene of interest

Common reporters.

Chloramphenicol acetyltransferase (CAT)

modifies chloramphenicol

Luciferase

Produces light (ATP-dependent)

Beta-galactosidase

hydrolyzes lactose analogs

The second two can be monitored in situ as well as in vitro

 

Southern blot used to analyze (genomic) DNA sequences


 


Total genomic DNA is purified and digested with restriction enzymes

This digest is electrophoresed on an agarose gel to separate the DNA fragments according to size. If you stain the DNA in the gel with ethidium bromide, a smear is usually seen since the digest may contain thousands of different sized fragments.

The gel is treated with Acid, then NaOH. This randomly nicks the DNA then denatures it.

The gel is then placed on moist blotting paper and a nitrocellulose filter is placed on top, followed by a large stack of dry paper towels (or a disposable diaper!).

The dry paper sucks buffer through the gel, carrying the DNA with it. Nicking the DNA (acid treatment above) allows the large fragments to transfer as efficiently as the small ones

 

Typically one uses a radiolabeled DNA or RNA probe to detect sequence of interest.

The temperature and buffer conditions used for hybridizing and washing the filters have dramatic effects on results.

Factors favoring hybridization (base-pairing of DNA strands):

Low temperature

High [salt]

Low [denaturant]

probe length

time

%GC content of probe

All can be factored to determine melting temperature of DNA duplex (Tm)

Tm=81+16.6log[Na+] Ð 0.4[%(G+C)] Ð 0.6 (% formamide) Ð 600/n Ð1.5(% mismatch)

where n is length of probe in bases

 

After autoradiography, a dark band should be seen which corresponds to a DNA restriction fragment homologous to the probe DNA. The size of the fragment can be determined by comparison with known DNA standards run on the same gel.

Southern are useful for optimizing probe conditions for library screening, for mapping genomic sequences flanking cloned regions and in forensic analysis.

 

Northern blot used to mRNA expression

Instead of DNA total RNA or polyA+ mRNA from tissues of interest is electrophoresed on a denaturing gel (agarose-formaldehyde or acrylamide-urea), separating RNAs by size. The gel is  blotted and probed as for a Southern Blot. Very useful for determining whether your DNA sequence is expressed as mRNA and how it is expressed.

Used to monitor regulation of mRNA levels:

Can isolate mRNA from different tissues

different times in development

cells treated differently in culture.

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.

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polymerase: usually thermostable polymerases and Òcycle sequencingÓ 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

 

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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 Òwild-typeÓ template

e.g. Òquick changeÓ strategy:


Cloning gene of interest:

Usually have to isolate gene of interest from a library of clones.

Library - representative collection of all (hopefully) DNA encoded in genome or cDNA of all mRNAs expressed in cells.

Genomic DNA - usually partially digested with restriction endo or mechanically sheared and ligated into vector.

--> overlapping clone sequences which span the genome, only a few of which contain your gene.

 

cDNA - copy of mRNA sequence made by reverse transcription of mRNA using retroviral reverse transcriptase. --> only exon sequences, much more convenient than genomic sequence for most genes (but may lose important regulatory sequences). Can make library of expressed genes for specific tissue.

 

For either type of library, you need a probe to detect the gene you want:

homologous DNA from related organism

cDNA for obtaining genomic clone (and vice versa)

antibody (need to use expression library)

oligonucleotide probe based on protein sequence

PCR product obtained from oligos based on shorter regions of homology

RFLP marker that maps near the gene you are interested in.

 

Need to know how many colonies (or phage plaques, which are usually easier to screen) are necessary to screen and be reasonably sure to get your clone.

to screen genomic DNA:

P= 1-(1-f)N or N=ln(1-P)/ln(1-f)

where f is size of average insert/size of genome, P is probability, and N= number of clones to screen

for 10kb insert you would need 2200 clones to screen the E. coli genome of 4640kb. This could be done on a single small petri plate

You would need 1.4 million of these inserts to screen the human genome

this would require nearly 30 large petri plates.

(expression libraries require even more: gene has to be in proper orientation and reading frame for detection with antibody)

 

The library is plated out and after plaques are seen, a nitrocellulose or nylon filter is overlaid onto the plate and marked to allow realignment of filter and plate later on. Filter is treated and probed just like a Southern blot

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Once the filters are probed, washed and dried, they are exposed to film. Black spots indicated putative clones of interest.

 

Once isolated, clones must be tested to determine whether they represent the gene of interest. One common test is the Northern blot to determine whether the DNA is homologous to a mRNA in the cell type of interest.

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