Introduction to

Cell biology

cells

tissues

organs

organisms

Nucleus, mitochondria, Golgi apparat, etc

Ribosomes, chromosomes, cytoskeleton, membranes, etc

proteins

aminoacids

nucleic acids

nucleotides

N-containing bases Ribose

monosaccharides

polysaccharides

phospolipids

Fatty acids, glycerol, cholin

triacylglycerols

Hierarchical organisation of the structure of living systems

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 theoryCell 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”

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."

Louis Pasteur

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

EM

Diagram

1 m 1 m

I. (BIO)CHEMICAL FOUNDATIONS

The most important groups of organic molecules: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:Amino acids:General chemical structure:General chemical structure:

NH2- -COOH

Peptide bound:Peptide bound:

NH2- -COOH + NH2- -COOH

NH2- -CONH- -COOH + H2O

20 different amino acids in unlimited amount in any possible 20 different amino acids in unlimited amount in any possible variations may form unlimited number of various peptide chainsvariations 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:

-helix-helix and -sheet -sheet ( L. Pauling, early 1950s)

In the rodlike -helix-helix the polypeptide backbone is folded into a spiral that is held in place by hydrogen bonds.

The sheet sheet consists of laterally packed strands strands (extended polypeptide structures). sheets are stabilized by hydrogen bonds between the strands.

The compact structure of the proteins is ensured by turns turns (compact, U-shaped elements stabilized by H-bonds) and loopsloops (long, loose bends) between the -helical and -sheet structures.

-Helix

strands

Loops and turns

An example: Ribonuclease

Tertiary and quaternary structure

Tertiary 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 structureQuaternary 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

Storage lipids (apolar)

Membrane lipids(polar)

Triacylglycerol

Phospholipids Glycolipids

Glycero-phospholipids

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.The most abundant biomolecules on the earth.

Essential components of foodstuff (sugar)Essential components of foodstuff (sugar)

Forms of occurence in living systems: Forms of occurence in living systems:

monosaccharides (e.g. glucose)monosaccharides (e.g. glucose)

oligosaccharides (e.g. saccharose, lactose)oligosaccharides (e.g. saccharose, lactose)

polysaccharides (e.g. glycogene, starch)polysaccharides (e.g. glycogene, starch)

Occurrence in complex macromolecules:Occurrence in complex macromolecules:

with lipidswith lipids (e.g.glycolipides)(e.g.glycolipides)

with proteins (glycoproteins and proteoglycans)with proteins (glycoproteins and proteoglycans)

within nucleic acids within nucleic acids (constituents of RNA and DNA)(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 nucleotidenucleotide is composed of

an organic base

a pentose (five-carbon sugar)

a phosphate group

SUGAR

PHOSPHATE

BASE

The base components of nucleic acids

N-containing (heterocyclic) ring molecules: purinespurines ( a pair of fused ring) and pyrimidinespyrimidines ( a single ring).

uracil

cytosine

thymine

adenine

guanine

cytosine (C), adenine (A) & guanine (G): in RNA and DNA thymine (T): in DNA uracil (U): in RNA

Chemical structure of nucleic acidsDNA or RNA strand formation: polymerization (condensation) of nucleotides, by forming phosphodiester bonds.

In RNA the sugar component is ribose (one OH more)

Nucleic acid sequence with one-letter codes:

e.g. A-C-T-T-C-G-G

beginning with 5’end

The RNA molecule is most often single-stranded. Intramolecular basepairs are forming frequently (e.g. tRNA), resulting in formation of secondary structure elements.

RNA

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

sugar-phosphate backbone

H-bonds

guanine

cytosine

adenine

thymine

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 complementarityBase-pair complementarity

A is paired with T

G is paired with C

Space-filling model of the DNA double

helix

Francis Harry Compton Crick

Institute of Molecular Biology Cambridge, United Kingdom

James Dewey Watson

Harvard University Cambridge, MA, USA

Nobel Prize 1962

„for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material”

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

Cell d

iv ision

DNA

DNA

RNA ProteinTrans- Trans-

criptioncription

Trans- Trans-

lationlation

Repli-Repli-cationcation

DNA – RNA – ProteinDNA – RNA – Protein

DNA stores the information

RNA is the messenger (sometimes stores information, sometimes acts as an enzyme)

Proteins are structural units and working molecules.

The central dogma of genetics

retroviruses

DNADNA4 Bases4 BasesA G C TA G C T

Organisation in tripletsOrganisation in triplets

RNARNA4 Bases4 Bases

A G C UA G C U1 triplet (codon) = 1 code word1 triplet (codon) = 1 code word

64 code words64 code words

ProteinProtein20 amino-20 amino-

acidsacids

More than one codon More than one codon for each amino acid.for each amino acid.

The code is redundantThe code is redundant..

The genetic code: organisation and transformation

The genetic code (RNA to amino acids)

The genetic code is (almost) universaluniversal: the meaning of each codon is the same in most known organism.

Unusual codon usage occurs in mitochondria, chloroplasts and several archaebacteria.

The genetic code is commaless! Thus:

5’___ GCUUGUUAACGAAUUA__ mRNA

Reading frames

__GCUUGUUAACGAAUUA

Ala--Cys--Leu--Arg--Ile

__GCUUGUUAACGAAUUA

Leu--Val--Tyr--Glu--Leu

GeneGene:The nucleotide sequence needed to produce a functionally competent „working molecule” (RNA or protein

Genome:Genome:The totality of the genes of a given organism.

I.4.2. Gene and genome

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

Genome Sequence Projects

• 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

The human genome

Gene expressionGene 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 transcriptiontranscription 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 translationtranslation 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.

1.4.3. Gene expression

Organization of genes in DNA in prokaryotes and eukaryotes

Prokaryotes: Protein-coding regions, organized in operonsoperons, are closely spaced along the DNA sequence.

Example: the lac operon of E. coli (Jacob and Monod, 1960s)

Y AZ

lac operonlac operonTranscription Transcription control regioncontrol region

P O

Promoter Promoter regionregion

Operator Operator regionregion

Eukaryotes: a considerable amount of DNA is untranslated Transcribed regions of most of the genes is composed of several exonsexons (translated from mRNA) and intronsintrons (eliminated from mRNA before translation).

Example: human beta globin gene:

50 90 130 222 850 126 132

Untranslated regions

Exons

Introns

Y AZP ORNA polymerase

start site for RNA synthesis transcription

Y AZ5’ 3’

translation

ZY

A

Proteins

Main features of gene expression in prokaryotes and eukaryotes

Polycistronic mRNAPolycistronic mRNA

start sites for protein synthesis

Prokaryotes

Example: lac operon

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 siteinitiation site on the duplex DNA and binds to it

2. The enzyme temporarily separates the two DNA strands

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.

3. De novo RNA synthesis begins by the binding of the first nucleotide by base pairing

1.4.3.2. Translation.

Participants:

• mRNAmRNA: source of the genetic information

• loaded tRNA: loaded tRNA: adaptormolecule, recognizing the codon and providing the corresponding amino acid.

• RibosomesRibosomes: the „machines” in which the proteins are produced on the basis of the genetic information provided by mRNA.

• Numerous other proteins serving as regulators: intiation- intiation- elongationelongation and terminationfactorsterminationfactors

• GTPGTP and ATPATP

Loaded tRNA

Base pairing

Function: to furnish the appropriate amino acid on the basis of the code on the mRNA. 3D (tertiary) structure

anticodon

TCG arm acceptor arm

D arm

anticod

on

arm

The ribosome

Small subunit Large subunit

mRNA

Exit of new peptide

Region of peptide synthesis

~50 proteins + 3 rRNAs ~ 33 proteins and 1 rRNA

Large subunit Mw ~ 2 800 000

Small subunit Mw ~ 1 400 000

Molecular components of ribosomes

The steps in translationA. 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 complexinitiation complex is ready to begin the synthesis

ATP and GTP is hydrolyzed and numerous proteins: „initiation factors”initiation factors” take part in these processes.

B. Elongation

P siteP site: outgoing site.

Direction of the ranslocation of ribosomes on mRNA: 5’ 5’ 3’ 3’

GTP is hydrolyzed, „elongation factors”elongation factors” take part

Elongation proceeds until STOP signal reached.

C. Termination

When the ribosome arrives to the stop codonstop codon (UAG) the translation is completed:

•• hydrolysis of peptidyl-tRNA on the ribosome

•• release of the completed polypeptide and the last tRNA

•• dissociation of the ribosomal subunits

Termination factors Termination factors play a role in the process. GTP is needed.

Free and ER-bound ribosomes

ER membrane

pool of ribosomal subunits in cytosol

mRNA encoding a cytosolic protein

mRNA encoding a protein targeted to ER

Peptide synthesis on ER-bound ribosomes

Posttranslational modification of proteins

After the completiton of translation numerous polypeptides and proteins undergo posttranslational modificationsposttranslational 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

5’

5’

3’

3’

Daughter duplex

Daughter duplex

Direction of fork

Parental DNA duplex

5’

3’ 5’

3’

Leading strand

Lagging strand

Okazaki fragments connected by DNA ligase

DNA polymerases carry out DNA synthesis on a DNA template, exclusively in 5’ to 3’ direction.

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 primerRNA primer, synthetised by the enzyme primase).

5’ 3’

Leading strand template

RNA primer, ~10 nucleotides long in eukaryotes

Leading strand: DNA synthesis is continuous

Leading strand

Lagging strand: DNA synthesis is discontinuous

5’ 3’

Lagging strand template

5’3’

replikation fork

New RNA primerOkazaki fragment (~200 nucleotides)

3’5’ 3’

New Okazaki fragment building up

3’5’ 3’

New Okazaki fragment finished

3’5’ 3’

Old primer erased and replaced by DNA

3’5’ 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 DNA repairrepair. DNA repair, similarly to replication, relies on base-pairing and involves several different pathways. If this process fails, permanent change – mutation 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.