Download Making RNA in other ways

15
• Central dogma
DNA--RNA--Proteins
• Retroviruses
– Classification
– Reverse transcription and the retroviral life
cycle
– Specific enzymatic functions
• RNA viruses
Making RNA in other ways
• Reverse transcription
• RNA replication
• Both mechanisms use RNA as the
information carrier rather than DNA
• Both are primarily viral mechanisms,
although reverse transcription also has
specialized functions in eukaryotic cells
Reverse transcription
• The central dogma of
molecular biology is DNA
to RNA to Protein
• Reverse transcription is a
reversal of the flow of that
information
• It uses RNA as the
information carrier and
produces a DNA
intermediate to function as
the template for further
synthesis of RNA
Reverse
transcriptase
• Synthesizes DNA from an
RNA template
• Made by retroviruses and
retrovirus derived genes
• RNA is not as stable as DNA
so retroviruses are often
fragile and easily destroyed by
exposure outside of a host
– Transmission is usually host to
host
• And it is sloppy (has low
fidelity) because it has no
proofreading capability
– As such, retroviruses suffer a
high mutation rate
• The mutations are usually
deleterious to the viruses
• However the mutation rate
results in a high adaptability to
adverse environments
• RT can be used to produce DNA copies of mRNA
– These are called cDNA
Reverse transcriptase
as a tool
• For copy DNA
• The copies can be cloned
• The advantage is that genes can be cloned without introns
– If an eukaryotic gene is to be expressed in a prokaryote, the
bacteria would not be able to splice the primary transcript RNA
from a eukaryotic gene
• But it would be able to make functional protein from cDNA because it lacks
introns
The retroviral genome
• The prototypical retrovirus is Rous Sarcoma Virus
– This causes solid tumors of fibroblastic origin and leukemias in
chickens
• It contains an oncogene known as src
• The genome moving 5’ to 3’
– R – repeat sequence
– PBS – primer binding site
• U5 + U3 + PBS + R = Long terminal repeat in duplex DNA
–
–
–
–
gag – the capsid genes (group antigen gene)
pol – enzymes: reverse transcriptase, protease, integrase
env – proteins in the envelope
src – the oncogene
Virion
• RSV is enveloped with a lipid
bilayer captured from its
previous host
• It binds to a cell and the
bilayer merges with the host
bilayer, releasing a capsid
containing two copies of its
RNA genome
– The capsid also contains copies
of reverse transcriptase
– Reverse transcriptase primes its
synthesis from a tRNA (also
captured from the previous
host) and makes an almost full
length copy of the RNA
genome
• The full length RNA-DNA
duplex is attacked by RNAse H
(part of reverse transcriptase)
resulting in degradation of the
RNA part of the heteroduplex
• Reverse transcriptase then
makes a full length copy of the
DNA
– Because of repeated sequences at
each end (long terminal repeats) no
sequence is lost
• The DNA can be cyclized and
sealed as a closed circle, or
remain as a linear form
• The viral DNA then integrates
into the host cell DNA and its
genes are expressed as though
they were normal cellular genes
Retroviral life
cycle II
• A host cellular tRNA primes
reverse transcriptase
• The resulting short
heteroduplex is degraded by
RNAse H
• The repeat sequence, now
DNA, rehybridizes to the
genome and primes RT again,
completing a single strand of
DNA
• RNAse H leaves a remaining
piece of genomic RNA to
prime synthesis of the second
DNA strand
– The tRNA is degraded, leaving
PBS sequences to hybridize and
prime synthesis again
– Synthesis is complete when
DNA priming fully extends the
strands in both directions
Reverse
Transcription
Retroviral life cycle
III
• Transcription yields full length
viral genomes and mRNA
• Gene expression yields selfassembling proteins that create
capsids containing retroviral
RNA, reverse transcriptase and
host cell tRNA primers
• The assembled capsid buds
through the membrane of the
cell where env proteins have
accumulated
• Thus an infected cell is not
necessarily killed, but serves
as a living factory for making
virus
Retroviral Gene
Expression
• Protease
• This protein cleaves the
product of retroviral
translation into fragments
– Only two or three mRNA’s
are transcribed
– They are translated into
polyproteins
• Polyproteins are a set of
different proteins synthesized
as a single protein
• Cleavage of the polyproteins
yields the mature final proteins
of the retrovirus
Retroviral integrase
• This acts to recombine the retroviral DNA with the host DNA
– The viral DS DNA is cleaved and the retroviral ends are crossed into the host
DNA
• Integration creates repeated host sequences at the site of integration
because the retroviral integrase makes staggered cuts at the site of
integration
• Integrase has no sequence specificity for the host sequences
– However it must recognize the viral genome
Retroviral diseases
• Retroviruses are classified into the
oncornaviruses and the lentiviruses
– Oncornaviruses are important causes of
veterinary cancers
• Leukemia in cats, mice, rats for example
• RSV is an oncornavirus causing sarcomas and
leukemias in chickens
– Oncornaviruses are not known to cause human
illness
• Lentiviruses are responsible for at least one
type of human cancer and AIDS
Oncornaviruses
Oncogenes
Simian sarcoma virus
erythroblastosis virus
Rat sarcoma virus (Harvey and Kirsten strains)
Murine leukemia virus
Myelocytomatosis virus
Myeloblastosis virus
Finkel osteosarcoma virus
• Oncornaviruses contain an
oncogene that is a mutant copy
of a normal cellular gene
• There is no advantage for a
virus to kill its host, however
there is an advantage for the
virus to force unregulated cell
division
– Cancer death may result from
infection, but the host will live a
relatively long time producing
virus
– When a cell is actively cycling, it
is making enzymes and
nucleotides in abundance in
preparation for cell division
• The virus needs these products
to make more virus
Lentiviruses
• The genomes are much more complex,
reflecting a finer regulation of gene
expression than the oncornaviruses
– HTLV I causes hairy cell leukemia, a rare
sexually transmitted disease
– HIV causes AIDS
• It is a mutant form of a simian virus that
made a species jump in Africa in the last
half of the 20th century
• The disease can be treated by using
deoxynucleotide analogs such as AZT, ddI
and ddC
– ddC is the same molecule as is used in DNA
sequencing
– The effect is the same as the sequencing
reaction, causing chain termination due to
the lack of a 3’ hydroxyl
• Protease inhibitors also interfere with the
cleavage of the polyproteins that are
produced by HIV transcription
• Together these treatments have had
dramatic effects on the survival of AIDS
patients
Resistance to drugs
• The activity of AZT depends on the ability of an enzyme to
mistakenly incorporate it instead of thymidine
• Random mutation of reverse transcriptase due to its inherent
error rate results in the chance occurrence of an RT that can
discriminate between the two nucleotides
– The same process works for any drug
• HIV that can survive exposure to these drugs are those that
are propagated
– Eventually the drugs become ineffective because only resistant
viruses replicate
– These become the viruses that are spread, so eventually the drugs
become ineffective throughout the population
– There may be constraints on the ability of an RT to mutate into a
form that can evade every deoxynucleotide analog
Retrotransposons
• These are retroviruses without an
external life cycle but sometimes
have a gag genes
– They make RT and integrase and have
LTR’s
– Retrotransposition scatters the
transposons indiscriminately throughout
the genome
• However some forms of
retrotransposition may be responsible for
the existence of introns
• Introns may become copied into DNA or
directly inserted into DNA as RNA
– The insertion is sequence specific
– So if cells fuse (as during fertilization) a
retrotransposon can copy an intron from
a gene on one chromosome and insert it
into the same gene at the same place on
the homologous chromosome
– This insures that the DNA sequence of
the two homologs is identical and
spreads the intron through the population
Telomerase – a reminder
• This is a reverse transcriptase, containing an RNA
template for the synthesis of DNA
• It binds to the ends of chromosomes and
synthesizes a short repeated sequence
• It then moves to the end of that sequence and
synthesizes it again, extending the lagging strand
end of DNA
• Telomerase is not present in most cells of the body
– However it is present in germ line cells, and is often
seen in cancer cells
RNA replicases
• These are exclusively viral
enzymes
– Many viruses have RNA
genomes that have no DNA
intermediate
• Influenza and Dengue fever
for example
• The genomes may be the
template or coding strand of
the viral mRNA, and may
also be double stranded
– They make an RNA
replicase that specifically
acts on their genome
– They have no proofreading
capability and resemble
RNA polymerase in their
activities
Origin of life
arguments FYI
• Initial experiments indicated that
adding simple salts, hydrogen
gas, water and methane and then
exposing them to heat and
electrical discharge in an
anaeorbic environment resulted
in the spontaneous synthesis of
amino acids and nucleotide bases
– This was an attempt to create a
primordial soup resembling
electrical discharge in a pre-biotic
ocean
• The abundance of adenine is
consistent with its current
importance in metabolism
throughout the biosphere
– ATP is always the energy currency
of a cell
• DNA is not known to be
catalytic
– Although formally it is because it
enters and leaves a reaction
unchanged
• How do you connect the
information in DNA to the
activities of protein?
WHY NOT RNA?
– DNA is the information
– Protein is the catalyst
• Both functions are present in
RNA
• The inference is that RNA was
in some way autocatalytic and
capable of synthesizing itself
• Evolution then produced more
stable forms of the
informational and catalytic
molecules