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

Reverse transcriptase as a tool

• RT can be used to produce DNA copies of mRNA– These are called cDNA

• 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

Retroviral life cycle II

• 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

Reverse Transcription

• 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

Retroviral life cycle III

• Transcription yields full length viral genomes and mRNA

• Gene expression yields self-assembling 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

OncornavirusesOncogenes

• 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

Simian sarcoma virus

erythroblastosis virus

Rat sarcoma virus (Harvey and Kirsten strains)

Murine leukemia virus

Myelocytomatosis virusMyeloblastosis virus

Finkel osteosarcoma 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

WHY NOT RNA?

• 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?– 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