Download The Cell Cycle

UNIT 5
Chapter 17: From Gene to Protein
Chapter 18: Microbial Models
Chapter 19: The Organization & Control of Eukaryotic Genomes
Chapter 20: DNA Technology
Introduction

The Central Dogma is the molecular “chain of
command” in a cell

DNA  RNA  proteins


Transcription: DNA used to make mRNA
Translation: mRNA used to make protein/polypeptide
Transcription: RNA Synthesis

RNA polymerase uses a template strand of DNA to
base pair with


Transcription includes: initiation, elongation,
termination
Initiation: RNA polymerase identifies template
strand by presence of promoter


TATA box
Transcription factors

RNA polymerase
base pairs RNA
nucleotides with
the template
strand

Uracil is used
in RNA rather
than thymine

Elongation: double helix
unwinds as RNA
polymerase adds
nucleotides


New RNA “peels off”
of the DNA as it
reforms the helix
A single gene can be
transcribed by many
RNA polymerase
molecules at once

Termination:
elongation
proceeds until a
terminator is
encountered


Primary
transcript is
released
In eukaryotes,
the transcript is
to be modified
RNA Processing

Before translation, the primary transcript undergoes
processing


5’ cap: added to the 5’ end to prevent digestion by
enzymes, also includes attachment site for ribosomes
Poly-A tail: added to the 3’ end to prevent digestion
by enzymes, also helps with exportation from nucleus

RNA splicing: non-coding sequences, introns, are
removed, leaving only exons

Spliceosomes made up of snRNPs facilitate splicing
of the exons
Translation: Polypeptide Synthesis

The newly created mRNA
(messenger RNA) enters the
cytoplasm and is attached to
a ribosome


Codons indicate which tRNA
is complimentary
tRNA (transfer RNA) carries
amino acids to the ribosome

Anti-codons correspond to
codons

Most codons correlate
with a specific amino
acid


Genetic code is
redundant but not
ambiguous
Start and stop codons

The genetic code is very
old and connects to our
scientific understanding
of evolution


It is almost universal
Foreign genes can be
expressed by
organisms

There are 61 codons, but only 45 types of tRNA (anticodons)



Base pairing rules are “relaxed” in the third position of the
codon/anti-codon
Called wobble: U can base pair with A or G
The ribosome is the site of translation



P site: holds tRNA with
growing polypeptide
A site: arrival site for next
tRNA
E site: site for discharging
tRNAs

Translation includes: initiation, elongation,
termination


Initiation and elongation require energy: GTP
Initiation: brings together mRNA, first amino
acid and two ribosomal subunits


First – small ribosomal subunit locates and attaches at
start codon
Second – tRNA carrying appropriate anti-codon (and
methionine) arrives and attaches to mRNA

Third – large ribosomal subunit arrives and covers the
tRNA at the P site (GTP required)

Initiation is now complete
P
A
E
met
UAC
5’ CGCCAUGCCUAGCACAUGACCUA
3’

Elongation: brings together remaining tRNAs in
order



First – the next tRNA will arrive and base pair with
the codon at the A site
Second – using GTP, a peptide bond is formed
between the new amino acid and the growing
polypeptide
Third – using GTP, the mRNA and tRNA are moved
in the 5’  3’ direction exactly three nucleotides
(translocation)
P
A
E
met
met
pro
UACGGA
5’ CGCCAUGCCUAGCACAUGACCUA
3’
P
A
E
met
met
pro
pro
UACGGA
UACGGA
GCCAUGCCUAGCACAUGACCUA
5’ CGCCAUGCCUAGCACAUGACCUA
3’
3’
P
A
E
met
pro
met
pro
ser
GGAUCG
GCCAUGCCUAGCACAUGACCUA
3’

Summary
of
elongation

Termination: ribosome encounters a stop codon

A release factor will base pair with the stop codon and
hydrolyze the polypeptide from the last tRNA

(Avg. protein translation: ~1 min)
Ribosomes

There are bound (on the rough endoplasmic
reticulum) and free (in the cytoplasm)
ribosomes



Bound: used to make proteins that will be
secreted from the cell
Free: used to make proteins that will stay in
the cytoplasm
Same mRNA can be translated by multiple
ribosomes – polyribosomes
Prokaryotes

Two major differences between eukaryotes
and prokaryotes

There is no RNA processing


What is transcribed IS the mRNA
Transcription and translation are coupled
END
Bacterial Genetic Material
Bacteria possess a single chromosome


Double-stranded, circular
4-6 million base pairs on average
Some bacteria carry plasmids with “non-crucial”
genes

Separate from chromosome, also circular
Variation in Bacterial Genetics
Bacteria can acquire new genes by one of three
methods: transformation, transduction,
conjugation

Transformation: bacteria take up foreign DNA and
incorporate it into their chromosome
Can also be plasmids

Transduction: phages act as vectors for bacterial
DNA
Accidental and rare

Conjugation: bacterial “sex” is the direct transfer of
genetic material between two bacteria
Requires an F factor (fertility) – gene that
allows for construction of a sex pilus

Hollow tube for transfer of plasmids
Most common type of shared plasmids =
antibiotic resistance
Regulation of Bacterial Genes
Bacteria have relatively simple control systems
for their genes called operons


Method for bacteria to turn on genes when needed
and off when not
Operons have three components: a promoter, an
operator, the gene(s) it controls
Promoter: site to which RNA poylmerase binds
Operator: site to which repressor protein binds

Repressor protein is always present in the cell
The lac operon is an example found in E. coli

Genes produce proteins/enzymes to digest lactose

No lactose:
Lactose:
Lactose binds
Repressor
canto
bind
repressor,
to operator
changing its conformation so it
cannot
bind
to operator
Prevents
RNA
polymerase from transcribing genes lacZ,
lacY, polymerase
RNA
lacA
can transcribe genes lacZ, lacY, lacA and
digest the lactose
END
Introduction
• Eukaryotic DNA is much more complex than that
of prokaryotes
• Little is known about expression
• Highly active area of research
• Genome is typically larger
• Cell specialization limits expression of genes
• Human genome possesses ~20K to 30K genes
• >97% of the genome is non-coding
• DNA is associated with MANY proteins
• Complex packaging can influence transcription
• Loose packing = frequent transcription; tight packing =
infrequent transcription
Gene Expression Controls
• Only a small portion of a
multicellular organism’s DNA
is actively transcribed in any
given cell
• Cellular differentiation makes
long-term control necessary
• 200 cell types, 1 genome
• Many levels of control exist to
regulate expression in
eukaryotes
Molecular Basis of Cancer
• Oncogenes are cancer-causing genes
• Arise from changes in a cell’s DNA (mutations)
• Chemical agents (carcinogens) or physical mutagens can
alter proto-oncogene function
• Mutations in tumor-suppressor genes can also cause
cancer
• Control adhesion of cells, inhibit cell cycle, repair damaged
DNA, initiate apoptosis
• Example of proto-oncogene includes p53
• Mutations to gene occur in 50% of all
cancers
• Nicknamed the “guardian angel of the genome”
• Damage to a cell’s DNA stimulates p53
expression
• Acts as a transcription factor for several other
genes
• Activates p21 gene which halts cell cycle
• Turns on genes involved in DNA repair
• If damage is irreparable, it turns on “suicide genes”
which causes cell death – apoptosis
Development of Cancer
• Usually, many mutations must occur for cancer to
develop
• Cancer is caused by the accumulation of mutations &
mutations occur throughout life  the longer we live,
the more chance of cancer
• Many malignant tumors have an active telomerase
gene
• Viruses (esp. retroviruses) account for 15% of
cancers
• They may donate oncogenes or disrupt tumorsuppressor genes or convert a proto-oncogene
END
Restriction Enzymes
• In nature, bacteria use restriction enzymes to cut
foreign DNA
• Restriction enzymes cut DNA at specific sites
• Enzymes identify a restriction site to cut at
• Restriction sites usually occur at many places in
a sequence of DNA
• Restriction sites may occur
at many locations, so the
enzyme will make many
cuts
• Often times, a staggered cut
is made, producing sticky
ends that can base pair with
its compliment
DNA Cloning Vectors
• Bacterial plasmids are used as cloning vectors
• DNA molecule that carries foreign DNA into a cell
• Bacteria can pass on their plasmids to daughter cells
• Less complex than eukaryotes, reproduce faster
• Cloning a human gene in bacteria steps
• Isolation of vector and gene of interest
• The vector is a plasmid
• Plasmid engineered to carry a gene for resistance to an antibiotic
• Insertion of gene of interest into vector
• Restriction enzymes used on both plasmid and
gene of interest to produce compatible sticky ends
• Gene and plasmid fragments mixed and DNA
ligase joins them together
• Introduction of recombinant vector into cells
• Bacteria are transformed by taking up plasmid
• Both recombinant and non-recombinant bacteria
are created
• Cloning of cells (and gene of interest)
• Bacteria are spread onto agar plates containing an
antibiotic
• Antibiotic ensures that only bacteria with the
plasmid will grow
• Transformed bacteria display “extra” trait
Complimentary DNA - cDNA
• RNA processing doesn’t occur in prokaryotes, so
it can be difficult to get them to express eukaryotic
DNA
• A fully processed mRNA is needed since its lacking
introns
• mRNA acts as a template for making DNA
• Reverse transcriptase used to make DNA from RNA
• Reverse transcriptase isolated from retroviruses
• Product is a cDNA molecule, DNA with no introns
compatible with bacterial DNA
• Creation of
cDNA
PCR
• The Polymerase Chain Reaction (PCR) can be
used to create billions of copies of a segment of
DNA in a few hours
• No cells are needed
• Nucleotides, primers, DNA polymerase added into
a test tube with our DNA to be copied
• PCR
• Special
DNA
Polymerase
is used
• Since 1985, PCR has had a huge impact on
biotechnology and DNA from a variety of sources
has been amplified
• A 40,000 year old frozen wooly mammoth
• TINY amounts of blood or semen (or other DNA
evidence) from crime scenes
• Embryonic cells for rapid diagnosis of genetic disorders
• Viral genes from difficult-to-detect viruses like HIV
END