The Central Dogma

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The Central Dogma
DNA replication
transcription
translation
DNA
RNA
Protein
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Prokaryotes Relatively Simple.
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Eukaryotes Not so Simple.
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Types of RNAs Produced in Cells
Types of RNAs
Functions
mRNAs messenger RNAs, code for
proteins rRNAs comprise ribosomes tRNAs adap
tors between mRNA and amino acids in
protein synthesis snRNAs splicing of
pre-mRNAs snoRNAs process and chemically
modify rRNAs MicroRNAs translation and
mRNA degradation Other non-coding telomere
synthesis, X-chromo. RNAs inactivation,
protein transport
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Types of RNA in a Eukaryotic cell

• mRNA
• rRNA
• tRNA
• primary transcript, precursor to mRNA, rRNA,
  tRNA, before processing and cleavage. Contains
  introns which may serve as ribozymes
• snRNA (small nuclear) plays structural and
  catalytic role in spliceosomes
• srpRNA component of signal recognition particle
  (recognises signal peptides on proteins targeted
  to the ER)
• si (small interfering) RNA and micro RNA
  involved in regulation of gene expression

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DNA-binding proteins often fit into the major
groove of the double helix
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Model of RNA PolII Preinitiation Complex
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Spatial Patterns of Gene Expression
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Eukaryotic Gene Structure

• Three basic types of eukaryotic genes
• Pol I genes (rRNA)
• Pol II genes (protein coding genes, some small
  RNAs)
• Pol III genes (small RNAs such as tRNAs

? Genes are the combination of a set of short
exons with very variable lengths of introns, ?
The distance between genes is often much larger
than the genes themselves.
The problem of the function of introns,
what is the role of the introns
is the great challenge in
molecular Biology.
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Regulation of Gene Expression

• Most genes in any type of cell are not expressed
• Spatial not every gene product needed in every
  cell type
• Temporal Different genes expressed at different
  times
• Environmental stimuli
• Hormones (internal chemical signals)

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Regulation of Gene Expression cntd.

• Level of expression of a gene usually refers to
  the amount of mRNA produced and rates of
  transcription
• Basic mechanism of regulation - similar in
  prokaryotes and eukaryotes expression of genes
  is activated/inhibited through proteins
  interactions with DNA

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GENE EXPRESSION IN EUKARYOTES An EXPLANATION In
Eukaryotes most genes have multiple overlapping
regulatory mechanisms that operate at more than
one level, from transcription through post
translation (See figure). There are no operons
in eukaryotes. However genes for related
functions are often regulated coordinately.
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Gene regulation in Eukaryotes

• Same principles as Prokaryotes, but additional
  levels of regulation
• chromatin packaging
• DNA methylation (imprinting)
• mRNA processing
• mRNA transport out of nucleus
• transcription factors proteins that bind to
  promoter region to enhance or prevent
  transcription
• gene silencing

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• Some promoter elements are required for
  transcription to begin.
• Eukaryotic protein coding genes contain both
  promoter elements and enhancer elements.
• Other promoter elements have a regulatory
  mechanism these are specialized for the gene
  they control, binding specific regulatory
  proteins that control expression of the gene.
• Specific regulatory proteins bind also to the
  enhancer elements and activate transcription
  through their interaction with protein bound to
  the promoter elements.

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• Chromosome regions that are transcriptionally
  active have loose DNA-protein structures than
  chromosome regions that are transcriptionally
  inactive, resulting in sensitivity of the DNA to
  digestion by Dnase.
• The promoter regions of active genes typically
  have an even looser DNA-protein structure,
  resulting in hypersensitivity to DNase.
• Transcriptionally active genes exhibit lower
  levels of DNA methylation.

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not all proteins are needed all the time in the
cell

• Cells need to respond to changing environment by
  regulating cell processes
• Most cell functions are performed by
  enzymes/proteins
• Two levels of regulation
• 1) amount of protein
• 2) activity level of protein
• Regulation is achieved in many different way

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Regulation of the amount of a protein in the cell

• Rate of transcription
• mRNA stability/rate of degradation
• Rate of translation
• Protein stability/rate of degradation
• -Post-translational modification
• glycosylation,
• phosphorylation,
• attachment of lipids/glycolipids
• -Binding to other proteins/substrate, etc

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DNA in Eukaryotes is not Naked

• DNA complexed with equal mass of protein
  chromatin
• Basic structural unit is nucleosome
• 147 bp DNA wrapped around a protein core
• Protein core comprised of histones (H2A, H2B,
  H3, H4)2
• Linker histone H1 joins nucleosomes together
• Histone tails subject to modifications that alter
  their highly basic charge
• Most common modification is acetylation of lysines

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1400 nm
700 nm
300 nm
30 nm fiber
30 nm
DNA wound around a cluster of histone molecules
Condensed chromosome
Scaffolding protein
Condensed chromatin
Extended chromatin
In Eukaryotes, DNA is packaged into chromosomes
with the aid of proteins called histones (see CR
Fig 19.2)
Packed nucleosomes
Histone
11 nm
Nucleosomes
2 nm
DNA double helix
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CHROMATIN STRUCTURE
Model of the chromatin fiber fromnucleosomes in
a high order helix.The disklike nucleosomes
associate toform a higher order helix with 6 to
8nucleosome units per turn. The inter- actions
stabilizing the fiber come from thehistone H1,
shown in red, thought to be at the center of the
helix, and possibly alsofrom the histone tail
regions.Adapted from F. Thoma, T. Koller andA.
Klug, J. Cell Biol. 83403 1979.
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Heterochromatin highly condensed, non-expressed
DNA Euchromatin less condensed, accessible to
transcription machinery, accessible to pancreatic
DNase I
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The inactive form of chromatin is the
heterochromatin (arrow) and the active form is
the euchromatin. In cells with a high level of
activity, the euchromatin is increased. The
nucleus in this cell contains a compact nucleolus
(N). Nucleoli are often large, pleomorphic and
multiple in neoplastic cells. Nucleoli are sites
of rRNA synthesis and are rich in RNA.
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Levels of Chromatin Packing
Compaction - The length of the total DNA content
of a human cell is nearly 1 meters. It must be
packed into a nucleus about 10-5 m in diameter.
DNA packs in several bundles chromosomes. The
length of the DNA in each of human chromosomes is
between 1.7 and 8.5 cm long. This is too long to
fit into a cell.
?
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2. The chromosomal DNA is packaged into a compact
structure with the help of specialized proteins
called histones.
The complex DNA plus histones
in eucaryotic cells is called chromatin.
?
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3. The fundamental packing unit is known as a
nucleosome. Each nucleosome is about 11nm in
diameter. The DNA double helix wraps around a
central core of eight histone protein molecules
(an octamer) to form a single nucleosome.
?
 
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• The Basis of Heredity
• All the information of cells is stored in their
  genetic material DNA
• The entire complement of genetic material of an
  organism is called its genome.
• In most prokaryotes, the genome consists of one
  large molecule of DNA (usually circular), called
  a chromosome.

• A chromosome is a molecule made of DNA and
  proteins.

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In eukaryotes, genes are NOT clustered in
operons. Eukaryotic genes often contain
non-coding introns ("intervening sequences")
interspersed among the coding regions (exons).
    During RNA processing, introns are removed
from RNA transcripts and the exons are spliced
together.
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Alternative RNA splicing in eukaryotes
(not available in bacteria) different
mRNA molecules are produced from the same primary
transcript,depending onwhich RNAsegments
aretreated as exons and which as introns.
H.A. Using this scheme explain the mechanism of
Alternative RNA splicing.
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Example of alternative splicing is the DSCAM gene
in Drosophila.
This
single gene contains some 116 exons of which 17
are retained in the final mRNA.

Some exons are always included others are
selected from an array.

Theoretically this
system is able to produce 38,016 different
proteins.

In fact, of 50 proteins synthesized
from mRNAs, 49 of them
turned out to be unique. Human about 25
thousand genes, We probably make at least 10
times that number of different proteins. More
than 50 of our genes produce pre-mRNAs that are
alternatively-spliced.
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? Repeated regions. A large part of the human
genome consists of repeated DNA. The (CCG)n and
(CAG)n repeats expansion can cause several
diseases in humans.
8 repeats 3 repeats
The number of repeats is highly variable among
individuals and the number of repeats is usually
different on the two pairs of chromosomes of one
individual). Analyzing the number of repeats
provides a highly sensitive measure of individual
identity and is the technique most often used for
forensic DNA typing.
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• The Basis of Heredity
• Prokaryotes have haploid genomes (i.e. one copy
  of each gene, located on the single circle
  chromosome).
• Eukaryotes have diploid genomes (i.e. there are
  two copies of each gene which are located on
  pairs of chromosomes one chromosome from each
  parental set)
  
• In eukaryotes, there are several chromosomes
  (always linear).

50 years ago new idea in GENETICS Genes could
be switch ON and OFF
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A gene includes the entire functional unit
coding DNA sequences, non-coding regulatory DNA
sequences, and introns. Genes can be as short as
1000 base pairs or as long as several hundred
thousand base pairs. The estimate for the
number of genes in humans are about
25,000 genes.
H.A. Do you understand the words non-coding
regulatory sequences and introns ? Explain.
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Chromosome Package" of genes and other DNA in
the nucleus of a cell. Different kinds of
organisms have different numbers of chromosomes.
Humans have 23 pairs of
chromosomes, 46 in all 44 autosomes
and two sex chromosomes.
Chromosomes
This figure schematically shows how DNA chains
fold into a compact form in chromosome.
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Chromosomes Bacterial chromosome - the DNA of a
typical prokaryote like E. coli
is contained in a single, large, supercoiled
circular DNA molecule.
Number of genes between 500-8000 Eukaryotic
chromosomes - The typical eukaryotic cell's
genome is divided into several chromosomes, each
of which contains a single, very large, linear
DNA molecule (of 107 to 109 bp in length).
Huge variability in genome sizes
The number of
eukaryotic chromosomes ranges from 1 (in an
Australian ant) to 190 (in a species of
butterfly).
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regulatory sequence - a DNA sequence responsible
for regulating gene expression, sites where
regulatory proteins such as, for example,
transcription factors bind preferentially.
H.A. transcription factors?
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Genome is defined as the sum of the genes of the
haploid cell.
A GENE one or more regions of a molecule DNA,
which code for a particular protein.
Bacterial gene
is a continuous region of DNA splits into
separated segments in genomic DNA
Eukaryotic gene
H.A. What is the difference between the
structures of Bacterial and Eukaryotic genes?
Exons (coding) segment Introns (not coding)
- the entire collection of proteins that are
encoded by the genome of an organism.
Proteome
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The genotype of an organism is the set of genes
possessed by an individual organism.
The phenotype is
observable physical characteristics of an
organism.