Download The Scientific Method in Biology

Questa è solamente una traccia; il significato di molte parole chiave
qui contenute deve essere espanso dallo studente con altre fonti.
A brief introduction
to DNA and
proteins.
The chemistry and biology of DNA
and proteins
Greg Gloor, Ph.D., Professor of Biochemistry
[email protected], 661-3526
Overall concepts
Modern biotechnology is built on the products of
manipulated DNA
Novel biotechnologies involve DNA directly or indirectly
(e.g. cloning, barcoding, genetic disease, DNA
computing)
DNA is an information medium
All information that flows within the cell, the body and
between generations is ultimately contained in DNA
DNA is an information medium
The role of DNA is to carry information …
within a cell
between generations of cells
between generations of organisms
DNA is an information medium
The role of DNA is to carry information …
It carries all the information required to build a new
organism
Including making a faithful copy of its own information
Information content
The double-helix structure of DNA (deoxyribonucleic acid) was discovered in 1954 by James Watson and
Francis Crick. A DNA molecule is composed of four different bases, guanine, thymine, cytosine and
adenine (G, T, C, and A, respectively) called nucleotide bases. The bases bind in pairs via a hydrogen bond,
and the pairs of bases form a long string, shaped in the form of a double helix. The pairs can only appear as
guanine opposite cytosine (G-C) or thymine opposite adenine (T-A), as sketched below:
TAC C G TAG G T CA. . .
| | | | | | | | | | | | |||
AT G G CAT C CAGT . . .
The string of base pairs forms a coded message, in which the bases are the characters of the "alphabet." If one of
the pairs of the string is known, then the other one is also known. This property is used during cell division,
when the helices unwind themselves and each half is copied. This copying activity can be considered
information transfer, but errors in the code may also occur.
If we consider a long string of, say, 100,000 bases, then the first "letter" may be either G, T, C, or A, or one of
four possibilities. For all 100,000 characters we then have 4 x 4 x 4 x ... 4 = 4100,000 = 2200,000 possible
strings of codes. If the probability of occurrence of all strings of codes is equal, then the probability of
finding a specific string is p = 2200,000. By Shannon's formula, the information content of the code
described by this molecule is therefore
I = -log2 p = -log2 2-200,000 = 200,000 (bits).
A DNA molecule of 100,000 base pairs has a length of approximately 500,000 Å and is 20 Å thick (1 Å = 10-10
m), which is impressive compared to the amount of space required to store a code of 100,000 bits in a
computer. A chromosome that contains on the order of 5 x 109 nucleotides, may code for 10 x 109 bits. For
the 23 chromosome pairs in the human genome, this would mean on the order of 5 x 1011 bits (equivalent
to about 60 gigabytes).
From: http://www.mihandbook.stanford.edu/handbook/home.htm
Information Flow
DNA
RNA
Protein
Information Flow
- major processes
DNA is in chromosomes
DNA is in chromosomes
- base pairs are the bits/letters
DNA is in chromosomes
- genes are information units
I cromosomi e il DNA



Ogni cellula del nostro corpo contiene una copia del nostro completo progetto
strutturale genetico costituito dai geni (detto anche patrimonio genetico o genoma)
Il progetto genetico è suddiviso, distribuito e compattato nelle cellule sotto forma di
cromosomi, in numero di 46 e costituiti da stringhe parziali di quei geni
Sia i cromosomi che i geni sono costituiti da DNA
■ I cromosomi e i geni sono contenuti nel nucleo delle cellule
(no nei globuli rossi, che non hanno un nucleo e quindi non
hanno geni e cromosomi) e, in piccola parte, anche nei
mitocondri, che hanno un ruolo energetico nella cellula



I cromosomi sono costituiti da un numero variabile di geni: alcuni contengono
diverse migliaia di geni, altri forse una o due migliaia
Non è ancora chiarito esattamente il criterio di ripartizione dei geni nei cromosomi
I geni sono sequenze di parole di tre caratteri presi dall’alfabeto A, T, G, C
Un sito che presenta una sintesi elementare di concetti base di genetica è:
http://www.genetics.com.au/pdf/factSheets/FS1.pdf
DNA is in chromosomes
- chromosomes package DNA



Chromosomes ensure
even division of copies of
DNA between daughter
cells
Packaging instructions
contained in centric
heterochromatin
Genes contained in
euchromatin
Information content of DNA




The sequence of the bases
along the DNA structure
contain the information
DNA has polarity and is
always read 5’ to 3’.
Thus 5’-GATA is different than
3’-GATA
All information in DNA is
redundant (AT bp and GC bp)
Quick Time™a nd a Gra phics de compr es sor a re ne eded to se e th is p ic tu re.
Information content of DNA
- read in the lab by sequencing
Bases are differentially labeled
(here by a fluorescent dye)
Each peak represents a different
base along the DNA sequence
As each base has one of 4
different fluorophores, the
unique sequence of the DNA is
determined
Information content of DNA
- DNA replication
DNA replication make a copy of
each original strand
Base-pairing controls the
specificity
DNA polymerase adds the DNA
DNA helicase unwinds the DNA
Primase makes the RNA primer
DNA topoisomerase untwists the
DNA
Copyright 1999 Access Excellence @ the National Health Museam
http://www.accessexcellence.org
Information content of DNA
- changes over time
About 1 base pair is changed
per genome per
replication cycle
1.5 x 109 bases per genome
1 x 1013 cells per body
Each cell in the adult has
~43 random base
differences with the
original genome
Information content of DNA
- two types of information
DNA information can be cis-acting
(intrinsic to the molecule) or transacting (extrinsic to the molecule
Cis-acting sequences control how the
DNA molecule interacts with the
cellular environment
Cis-acting sequences have specific names
(centromere, telomere)
Trans-acting sequences ultimately
produce the intracellular environment
(proteins, carbohydrates, small
molecules, etc)
Trans-acting sequences are usually called
genes
QuickTime™ and a Cine pak decomp ress or are nee ded to s ee this picture.
Information content of DNA
- cis-information
Controls the behavior of the chromosome
Centric heterochromatin controls how the
cell divides chromosomes during cell
division
Telomeric heterochromatin caps the ends
of the chromosome(which is a linear
molecule of DNA)
QuickTime™ and a Cine pak decomp ress or are nee ded to s ee this picture.
Information content of DNA
- is transcribed into RNA
RNA is a copy of the information
from one of the DNA strands
Process is called transcription
Primary point of control of gross
levels of gene products
Base-pairing controls the specificity
RNA is unstable and transient
compared to DNA
RNA can be used directly or be an
intermediate for protein
production
QuickTime™ and a Cine pak decomp ress or are nee ded to s ee this picture.
Information content of DNA
- a digression into proteins
Proteins perform most of the actual
work in the cell (structure,
enzymes, etc)
Proteins are linear polymers of
amino acids
There are 20 naturally occurring
amino acids commonly found in
proteins
The sequence of a protein
corresponds to the sequence of
the DNA/RNA that encodes it
Proteins fold spontaneously into a
the proper 3-D structure
Information content of DNA
- is usually translated into protein via the RNA intermediate
Information in RNA contained in triplets
of bases (codons)
Codons are non-overlapping and abutting
Each codon contains the information
required to include one and only one
amino acid at a position in a protein
The genetic code is universal (except for
a few small differences in some very
weird organisms)
Knowing the DNA or RNA sequence of a
chromosome, then allows us to
determine the sequences of all the
proteins which could be made by the
information on that chromosome
Terms

An enzyme is a biological catalyst, almost always
composed of one or more proteins (although RNA is also
capable of being an enzyme).
Chemical Reaction
Converts one or more reactants
(H2O2) into one or more products
(H2O, O2)
 Requires energy to start the
reaction
 Products have less energy than
reactants
 Proceeds through a transition
state, where the chemical bonds in
the reactants are stretched to their
limit without being broken
Source:http://explanation-guide.info/meaning/Activation-energy.html
Catalyst




Reduces the activation energy
Therefore the reaction proceeds
more rapidly, with less input
energy
The catalyst is not consumed
One catalyst can thus enable
multiple instances of the same
reaction
Source:http://explanation-guide.info/meaning/Activation-energy.html
Enzyme





An enzyme is a biological catalyst
Generally enzymes are proteins, but
sometimes they are composed of
RNA or RNA and protein
Only a small portion of the enzyme
(shown as balls or sticks) is involved
in catalysis of the reactant (shown in
red)
The remainder of the protein (dots)
holds the catalytic residues in the
proper 3-D place
Catalase converts hydrogen peroxide
to water and oxygen
Review of Information Flow
Four levels of protein structure




Primary - sequence
Secondary - initial fold
Tertiary - 3-D folding
Quaternary - interactions
between proteins
The peptide bond





Alpha helices and beta sheets predominate
secondary structures
They are composed of peptide bonds that repeat
their bond angles for several residues
Peptide backbones are free to rotate around the
bonds shown in blue
This rotation is constrained by the R groups (the
20 possible aa side chains)
Each peptide bond can assume one of 8 possible
conformations (on average) so the number of
possible protein conformations is (n-1)8, where
n is the number of residues in the protein
Secondary structure



Alpha helices and beta
sheets predominate
secondary structures
They are composed of
peptide bonds that repeat
their bond angles for
several residues
Ionic, hydrogen, van der
Waals and hydrophobic
interactions all help to
stabilize these structures
Tertiary structure



Alpha helices and beta sheets predominate
tertiary structures
Protein assumes its characteristic 3-D
shape by the secondary structural elements
folding on themselves
Ionic, hydrogen, van der Waals and
hydrophobic interactions all help to
stabilize these structures
Quaternary structure


Proteins fit together the secondary and
tertiary structural elements folding on
themselves
Ionic, hydrogen, van der Waals and
hydrophobic interactions all help to
stabilize these structures
What governs activity?
Chemical Equilibrium of a DNA binding
protein
Kd = 1E-10 M/L
[Protein][Ligand]
Kd = ––––––––––––––––
[Protein:Ligand]
Concentrations are in moles/liter
Example bacterial cell volume = 1.5E-15 L
1 molecule of DNA = 1/6.02E23 moles =
1.66E-24 moles
1 molecule per cell =
1.66E-24 moles / 1.5E-15 litres = 1.1E-9 M/L
5 molecules protein / cell = 5.5E-9 M/L
98% of the DNA sites are occupied under this
condition
Bound Ligand [Protein]
––––––––––– = –––––––––––
Free Ligand
Kd + [Protein]
B
–– = .982
F
(thanks to Dr. Brian Shilton)
This means …


That reactions in the cell that depend on proteins
are almost never fully on or fully off.
Most operations in the cell operate as an analogue
machine in intermediate stages of activation
Synthesis, Folding, Degradation


Synthesis rates account for the relative amount of
protein made at any one time
Depends upon the amount of mRNA made from
the gene and upon the rate that mRNA is
translated
Synthesis, Folding, Degradation


Most proteins assume their final structure with the
help of chaperones (proteins that aid folding)
Chaperones increase the speed of folding and alert
degradation pathways to proteins that cannot fold
Synthesis, Folding, Degradation



Pathways for degradation include lysosomes and
ubiquitin-dependent pathway
Proteins are degraded to peptides and amino acids
extracellularly (digestive tract, etc) as well
Some modifications, such as acetylation or
glycosylation, increase the protein’s life span