Introduction to
Cell biology
Hierarchical organisation of the structure of
living systems
organisms
organs
tissues
cells
Nucleus, mitochondria, Golgi apparat, etc
Ribosomes, chromosomes, cytoskeleton, membranes, etc
proteins
aminoacids
nucleic acids
polysaccharides
nucleotides
monosaccharides
N-containing bases
Ribose
triacylglycerols
phospolipids
Fatty acids,
glycerol, cholin
Cells as seen before the cell theory
Anton van Leeuwenhoek, XVII. century:
algae, bacteria, sperm cells, etc.
Robert Hooke 1665: „cell”: unit in
dead samples of cork.
The cell theory
Cell as the central unit of biological organization
• Cells are the basic units of life.
• All living organisms are made up of cells.
• Only living cells can produce new cells.
Matthias Schleiden 1838
Theodor Schwann 1835
plants are made up of cells
animals are made up of cells
Rudolf Virchow
1858:
„Every animal appears
as the sum of vital units,
each of which bears in itself
the complete characteristics
of life”
Louis Pasteur
1865 : „Spontaneous generation” of life ruled out experimentally
„There is now no circumstance known in which it can be affirmed
that microscopic beings came into the world without germs, without
parents similar to themselves."
Tranzitions from non-living towards
living: I. Prions: molecules resembling
ion channels,
causing serious illnesses
Tranzitions from non-living towards living:
II. Viruses
Viruses have no metabolism and can not reproduce by
themselves. They contain genetic material (either RNA or DNA)
and proteins. After infection they use the machinery of the host
cell to produce more viruses.
Highly simplified
structure of a virus
The HIV virus
Prokaryotic and eukaryotic cells
Diagram
EM
1 mm
1 mm
I. (BIO)CHEMICAL FOUNDATIONS
The most important groups of organic molecules:
Proteins composed of amino acids
Lipids composed of glycerol and fatty acids
Carbohydrates: mono-, oligo- and polysaccharides
Nucleic acids: DNA, RNAs
I./1. PROTEINS
Classification of proteins:
• Enzymes
• Receptors
• Transport proteins
• Storage proteins (casein in milk, ferritin /iron/)
• Contractile proteins
• Structural proteins
• Immune proteins
• Regulatory proteins
• Others (e.g. antifreeze proteins)
Amino acids:
General chemical structure:
NH2-kkkkk-COOH
Peptide bound:
NH2-kkkkk-COOH + NH2-kkkkk-COOH
i
NH2-kkkkk-CONH-kkkkk-COOH + H2O
20 different amino acids in unlimited amount in any possible
variations may form unlimited number of various peptide chains
Primary structure
Primary structure or sequence: linear arrangement of the
amino acids that constitute the
polypeptide chain
Sequencing:
to determine the order of amino acids of a protein.
Sequence motive:
a specific amino acid arrangement that
appears in several different proteins and play
the same role in these proteins.
Examples:
DNA binding motive
signal sequence (transport of the protein to a
given organelle)
sequence for phosphorylation
ligand-binding sequences (e.g. ATP, growth
hormons)
Secondary structure
Local organisation (folding) of parts of a polypeptide chain.
Most important secondary structure elements:
a-helix and b-sheet ( L. Pauling, early 1950s)
In the rodlike a-helix the polypeptide backbone is folded into a
spiral that is held in place by hydrogen bonds.
The b sheet consists of laterally packed b strands (extended
polypeptide structures). b sheets are stabilized by hydrogen
bonds between the strands.
The compact structure of the proteins is ensured by turns (compact,
U-shaped elements stabilized by H-bonds) and loops (long, loose
bends) between the a-helical and b-sheet structures.
An example: Ribonuclease
a-Helix
b strands
Loops and turns
Tertiary and quaternary structure
Tertiary structure: Three-dimensional arrangement of all amino
acids, which results in mainly from hydrophobic interactions
between nonpolar amino acid side-chains. These interactions
hold helices, strands and coils together. The highest level of
organisation for monomeric proteins.
Quaternary structure: number and relative positions of subunits
in multimeric proteins.
Determination of the three-dimensional structure of proteins:
x-ray crystallography
nuclear magnetic resonance (NMR)
An example: Haemoglobin
I./2. LIPIDS AND THEIR COMPONENTS
Membrane lipids
(polar)
Storage lipids
(apolar)
Phospholipids
Triacylglycerol
Glycerophospholipids
Glycolipids
Sphingolipids
Triacylglycerols
Serve for storage (lipid droplets in fat cells) and isolation.
Membrane lipids
Cholesterol
In addition to the phospholipids, it occurs in biological
membranes – exclusively in eukaryotes.
Stabilizes the membranes.
I./3. CARBOHYDRATES
The most abundant biomolecules on the earth.
Essential components of foodstuff (sugar)
Forms of occurence in living systems:
monosaccharides (e.g. glucose)
oligosaccharides (e.g. saccharose, lactose)
polysaccharides (e.g. glycogene, starch)
Occurrence in complex macromolecules:
with lipids
(e.g.glycolipides)
with proteins (glycoproteins and proteoglycans)
within nucleic acids
(constituents of RNA and DNA)
Some monosaccharides
Glycogene: polysaccharide
I./4. NUCLEIC ACIDS
Nucleic acids are the information-storing molecules of the cells.
They are linear polymers of nucleotides connected by
phosphodiester bonds.
A nucleotide is composed of
BASE
an organic base
a pentose (five-carbon sugar)
PHOSPHATE
a phosphate group
SUGAR
The base components of nucleic acids
N-containing (heterocyclic) ring molecules:
purines ( a pair of fused ring) and
pyrimidines ( a single ring).
adenine
uracil
cytosine
thymine
guanine
cytosine (C), adenine (A) & guanine (G): in RNA and DNA
thymine (T): in DNA
uracil (U): in RNA
Chemical structure of nucleic acids
DNA or RNA strand formation: polymerization (condensation)
of nucleotides, by forming phosphodiester bonds.
Nucleic acid sequence with
one-letter codes:
e.g. A-C-T-T-C-G-G
beginning with 5’end
In RNA the sugar component is
ribose (one OH more)
RNA
The RNA molecule is most often single-stranded.
Intramolecular basepairs are forming frequently (e.g. tRNA),
resulting in formation of secondary structure elements.
Further organization of secondary structures
lead to the appearance of tertiary structure.
• A considerable fraction of RNA occurs in great
complexes together with proteins (e.g. ribosomes)
• RNA can have catalytic activity (ribozymes).
• RNA is the genetic material in several viruses (polio,
influenza, rota, HIV, etc).
DNA: its native state is a righthanded double
helix of two antiparallel chains
The bases of the two chains ( one running 5’ 3’, the other one 3’5’)
are held in precise register by H-bonds.
Base-pair complementarity
thymine
cytosine
G is paired with C
H-bonds
adenine
sugar-phosphate backbone
A is paired with T
guanine
Space-filling
model of the
DNA double
helix
Nobel Prize 1962
„for their discoveries
concerning the
molecular structure
of nucleic acids and
its significance for
information transfer
in living material”
James Dewey
Watson
Francis Harry Compton
Crick
Harvard University
Cambridge, MA, USA
Institute of Molecular Biology
Cambridge, United Kingdom
General principles of nucleic acid polymerization
1. Both DNA and RNA chains are produced in cells by
copying a preexisting DNA strand (template) according to
the rules of Watson-Crick DNA pairing /A-T, G-C, A-U/.
2. Nucleic acid growth is in one direction: from the 5’
(phosphate) end to the 3’ (hydroxyl) end.
3. Special enzymes (polymerases) are necessary to produce
DNA or RNA.
4. DNA double helix synthesis by base-pair copying requires
the unwinding of the original duplex. A single stranded
region (growing fork) is formed.
I.4.1. Cellular processes involving nucleic acids
Gene expression
Trans-
DNA
RNA
Translation
cription
Replication
DNA
Protein
The central dogma of genetics
DNA – RNA – Protein
retroviruses
DNA stores the information
RNA is the messenger (sometimes stores
information, sometimes acts as an enzyme)
Proteins are structural units and working
molecules.
The genetic code: organisation and
transformation
DNA
4 Bases
AGCT
Organisation in triplets
RNA
4 Bases
AGCU
1 triplet (codon) = 1 code word
64 code words
Protein
20 aminoacids
More than one codon
for each amino acid.
The code is redundant.
The genetic code (RNA to amino acids)
The genetic code is (almost)
universal: the meaning of
each codon is the same in
most known organism.
Unusual codon usage occurs
in mitochondria,
chloroplasts and several
archaebacteria.
Reading frames
The genetic code is commaless! Thus:
5’___ GCUUGUUAACGAAUUA__
__GCUUGUUAACGAAUUA
Ala--Cys--Leu--Arg--Ile
mRNA
__GCUUGUUAACGAAUUA
Leu--Val--Tyr--Glu--Leu
I.4.2. Gene and genome
Gene:
The nucleotide sequence needed to produce a
functionally competent „working molecule” (RNA or
protein
Genome:
The totality of the genes of a given organism.
Genome Sequence Projects
Since 1995 the following complete genom sequences became
available:
Prokaryotes:
More than 30 Bakterial species (several disease-causing ones), some
Archaebakteria
Eukaryotes:
Saccharomyces cerevisae (baker’s yeast)
Caenorhabditis elegans (worm)
Drosophila melanogaster (fruitfly)
Arabidopsis thaliana (plant)
Mus musculus (mouse)
Homo sapiens
The human genome
• the sequence of the human genom contains
3,3 billion bases, organised in 24 chromosomes
(22, X,Y)
• 30 000 to 40 000 genes
• 233 genes are evidently of bacterial origin
• 98 % of the sequence is „nonfunctional”
• the genetic identity of the human beings is
99.9 %
Nature, 15. February 2001/Science, 16. February 2001
1.4.3. Gene expression
Gene expression: the entire cellular process whereby the
information encoded in a particular gene is decoded to a
particular protein.
Molecular processes involved in gene expression: transcription
und translation.
During transcription an RNA (messenger RNA, mRNA) is
synthesized, which contains the genetic information of the DNA
as a complementary sequence. The procedure is catalyzed by
DNA dependent RNA polymerases.
During translation the nucleotide sequence of the mRNA is
converted to amino acid sequence of a protein. Besides the
mRNA, ribosomes and tRNAs numerous enzymes and
regulator proteins play important roles in this procedure.
Organization of genes in DNA in prokaryotes and
eukaryotes
Prokaryotes: Protein-coding regions, organized in operons,
are closely spaced along the DNA sequence.
Example: the lac operon of E. coli (Jacob and Monod, 1960s)
lac operon
Transcription
control region
P
Promoter
region
O
Operator
region
Z
Y
A
Eukaryotes: a considerable amount of DNA is untranslated
Transcribed regions of most of the genes is composed of several
exons (translated from mRNA) and introns (eliminated from
mRNA before translation).
Example: human beta globin gene:
50
90
130
222
Untranslated regions
Exons
Introns
850
126
132
Main features of gene expression in prokaryotes
and eukaryotes
Prokaryotes
Example: lac operon
RNA polymerase
P
O
Z
Y
start site for RNA
synthesis
5’
Z
A
transcription
3’
Polycistronic mRNA
Y
A
start sites for
protein synthesis
translation
Proteins
Z
Y
A
Eukaryotes:
• Trancription occurs in the
nucleus, translation in the
cytoplasm.
• Primary RNAs undergo
processing within the nucleus:
 addition of 5’cap
 polyadenilation
 splicing (removal of introns)
• mRNAs are monocistronic.
• Besides the nucleus, DNA
occurs also in mitochondria and
chloroplasts.
1.4.3.1. Transcription
Catalyzed by DNA dependent RNA polymerase.
Steps of the procedure:
1. the RNA polymerase finds an appropriate initiation site on the duplex
DNA and binds to it
2. The enzyme temporarily separates the two DNA strands
3. De novo RNA synthesis begins by the binding of the first nucleotide
by base pairing
4. The second nucleotide binds by base paring. The enzyme catalyses the
linkage of the two nucleotides (PPP remains at the 5’ end, PPi is split off
from the second nucleotide).
5. The third nucleotide binds and the enzyme links it to the existing
dinucleotide. The procedure continues until the STOP codon.
1.4.3.2. Translation.
Participants:
• mRNA: source of the genetic information
• loaded tRNA: adaptormolecule, recognizing the codon and
providing the corresponding amino acid.
• Ribosomes: the „machines” in which the proteins are produced
on the basis of the genetic information provided by mRNA.
• Numerous other proteins serving as regulators: intiationelongation and terminationfactors
• GTP and ATP
Loaded tRNA
Function: to furnish the appropriate
amino acid on the basis of the code on
the mRNA.
3D (tertiary) structure
TCG arm
acceptor arm
anticodon
Base
pairing
The ribosome
mRNA
Region of peptide
synthesis
Large subunit
Small subunit
Exit of new
peptide
Molecular components of ribosomes
Large subunit
Small subunit
Mw ~ 2 800 000
Mw ~ 1 400 000
~50 proteins + 3 rRNAs
~ 33 proteins and 1 rRNA
The steps in translation
A. Initiation
a.) a „partial” initiation complex forms:
Met-tRNAmet binds to the small ribosomal
subunit
b.) the above complex binds to the initiation
site on mRNA: AUG (codon of Met)
c.) by binding of the large subunit the
initiation complex is ready to begin the
synthesis
ATP and GTP is hydrolyzed and numerous proteins: „initiation factors”
take part in these processes.
B. Elongation
P site: outgoing site.
Direction of the ranslocation of ribosomes on mRNA: 5’  3’
GTP is hydrolyzed, „elongation factors” take part
Elongation proceeds until STOP signal reached.
C. Termination
When the ribosome arrives to the stop codon (UAG) the
translation is completed:
• hydrolysis of peptidyltRNA on the ribosome
• release of the completed
polypeptide and the last
tRNA
• dissociation of the
ribosomal subunits
Termination factors play a role in the process.
GTP is needed.
Free and ER-bound ribosomes
mRNA encoding a
cytosolic protein
pool of ribosomal
subunits in cytosol
mRNA encoding a
protein targeted to ER
ER membrane
Peptide synthesis on ER-bound ribosomes
Posttranslational modification of proteins
After the completiton of translation numerous polypeptides
and proteins undergo posttranslational modifications. These
modifications can influence their structure and function.
Most important posttranslational modifications:
• specific proteolysis
• removal of the first Met
• glycosylation
• phosphorylation
1.4.4. DNA replication
Semiconservative replication: every double helix contains a parent
strand and a newly synthetised one.
Parent
First
generation
Second
generation
Synthesis of the complementary daughter DNA
strands
DNA polymerases carry out DNA synthesis on a DNA
template, exclusively in 5’ to 3’ direction.
3’
5’
5’
3’
Leading strand
Parental DNA duplex
3’
5’
Direction of fork
Okazaki fragments
connected by DNA
ligase
3’ Lagging strand
5’
DNA polymerases are unable to initiate de novo DNA
synthesis, but can add nucleotides to the 3’end of preexisting
RNA or DNA strands (RNA primer, synthetised by the enzyme
primase).
Leading strand: DNA synthesis is continuous
Leading strand template
5’
Leading strand
3’
RNA primer, ~10 nucleotides
long in eukaryotes
Lagging strand: DNA synthesis is discontinuous
Lagging strand template
replikation fork
5’
3’
Okazaki fragment
(~200 nucleotides)
5’
3’
5’
3’
5’
3’
5’
3’
5’
3’
New RNA primer
3’
New Okazaki fragment building up
3’
New Okazaki fragment finished
3’
Old primer erased and replaced by DNA
3’
Nick sealing by DNA ligase joins new
Okazaki fragment to the growing strand
DNA repair, mutations
Maintainig genetic stability requires accurate mechanism
of replication as well as repair of lesions that occur
continually in DNA. Most spontaneous changes are
immediately corrected by the complex process of DNA
repair. DNA repair, similarly to replication, relies on basepairing and involves several different pathways. If this
process fails, permanent change – mutation – occurs in
DNA. Mutations in vital positions of the DNA sequence
destroy the organism, others might cause advantageous
modifications in the gene products, contributing to the
driving force of the evolution.