11-Biochemistry-Lecture11.pdf

Lecture 11 RNA-dependent synthesis of RNA and DNA Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 26: p 988-995 RNA METABOLISM - DNA-Dependent Synthesis of RNA ( see Lecture 9) - Post-transcriptional processing of RNA (See Lecture, 10) - RNA-Depende...

Lecture 11 RNA-dependent synthesis of RNA and DNA Additional material for this lecture may be found in: § Lehninger’s Biochemistry (8th ed), chapter 26: p 988-995 RNA METABOLISM - DNA-Dependent Synthesis of RNA ( see Lecture 9) - Post-transcriptional processing of RNA (See Lecture, 10) - RNA-Dependent Synthesis of RNA and DNA (This lecture, 11) Learning objectives: – RNA-dependent synthesis of RNA and DNA Retroviruses and reverse transcriptase Telomerase RNA-DEPENDENT SYNTHESIS OF RNA AND DNA RNA-DEPENDENT SYNTHESIS OF RNA AND DNA Extension of the central dogma : RNA-dependent synthesis of RNA and DNA Retroviral infection of a mammalian cell and integration of the retrovirus into the host chromosome Viral particles entering the host cell carry viral reverse transcriptase and a cellular tRNA (picked up from a former host cell) already base-paired to the viral RNA. The tRNA facilitates immediate conversion of viral RNA to doublestranded DNA by the action of reverse transcriptase. Once converted to double-stranded DNA, the DNA enters the nucleus and is integrated into the host genome. The integration is catalyzed by a virally encoded integrase. On transcription and translation of the integrated viral DNA, new viruses are formed and released by cell lysis (right). In the viruses, the viral RNA is enclosed by capsid proteins called Gag and outer envelope proteins called Env. Additional viral proteins (reverse transcriptase, integrase, and a viral protease needed for posttranslational processing of viral proteins) are packaged within the virus particle with the RNA. RETROVIRUSES CAN MAKE DNA FROM RNA Retroviruses have genomes of ssRNA and the enzyme Reverse Transcriptase (RT) – Virus enters host cell – RT makes DNA from the RNA Then degrades the RNA from the DNARNA hybrid and replaces it with DNA DNA can then be incorporated into host DNA REVERSE TRANSCRIPTASES CATALYZE THREE REACTIONS 1) RNA-dependent DNA synthesis 2) RNA degradation 3) DNA-dependent DNA synthesis Contain Zn2+ Use a primer of tRNA Lack 3’ à 5’-proofreading (like RNA Pol) - Makes RT error-prone - Explains high rate of virus mutation/evolution HIV RT RETROVIRUSES TYPICALLY CONTAIN THREE GENES PLUS A LONG TERMINAL REPEAT gag (group associated antigen) – Encodes a long polypeptide that is cleaved into six smaller proteins that make up viral core pol – Encodes protease that cleaves the long polypeptide, reverse transcriptase, and an integrase to insert DNA into host genome env – Encodes viral envelope Long terminal repeat (LTR) sequences facilitates integration of virus genome into host DNA STRUCTURE AND GENE PRODUCTS OF AN INTEGRATED RETROVIRAL GENOME and splicing - The long terminal repeats (LTRs) have sequences needed for the regulation and initiation of transcription. The sequence denoted ψ is required for packaging of retroviral RNAs into mature viral particles. - Transcription of the retroviral DNA produces a primary transcript encompassing the gag, pol, and env genes. - Splicing of the primary transcript yields an mRNA derived largely from the env gene, which is also translated into a polyprotein, then cleaved to generate viral envelope proteins. - Translation produces a polyprotein, a single long polypeptide derived from the gag and pol genes, which is cleaved into six distinct proteins. SOME RETROVIRUSES CAUSE CANCER Some retroviruses contain an oncogene. – Example: Rous sarcoma virus has the src gene Src for sarcoma, a cancer of bone, fat, muscle, etc. (vs. cancer of epithelial cell origin) Encodes a non-receptor tyrosine kinase, an enzyme that affects cell division Rous sarcoma virus genome. The src gene encodes a tyrosine kinase, one of a class of enzymes that function in systems affecting cell division, cell-cell interactions, and intercellular communication (Chapter 12). The same gene is found in chicken DNA (the usual host for this virus) and in the genomes of many other eukaryotes, including humans. When associated with the Rous sarcoma virus, this oncogene is often expressed at abnormally high levels, contributing to unregulated cell division and cancer. HIV RETROVIRUS CAUSES AIDS HIV genome has genes for killing host (mostly T lymphocytes) – Results in suppression of immune system HIV-encoded reverse transcriptase is unusually error-prone – Complicates push for vaccine – At least one error per replication, so potentially no two viral RNAs alike The genome of HIV, the virus that causes AIDS. In addition to the typical retroviral genes, HIV contains several small genes with a variety of functions (not identified here and not all known). Some of these genes overlap. Alternative splicing mechanisms produce many different proteins from this small (9.7 x 103 nucleotides) genome. PHARMACEUTICAL TARGETS FOR HIV (ANTIRETROVIRAL DRUGS) Reverse transcriptase inhibitors – nucleotide or nucleoside analogs – drug names ending in “dine” or “sine”: zidovudine (AZT), didanosine (Videx), and so on Protease inhibitors – since proteases are used in cleaving proteins for packaging into new viral particles – drug names ending in “avir”: indinavir, saquinavir, and so on TELOMERASE: A SPECIFIC REVERSE TRANSCRIPTASE that extends the ends of linear chromosomes The ends of a linear chromosome are not readily replicated by DNA polymerase – (since beyond the end, no template is available for pairing with an RNA primer) Without a special mechanism for replicating the ends, chromosomes will be shortened in each cell generation (loss of information) Telomerase solves this problem by adding telomeres to chromosome ends TELOMERES Are structures at the ends of eukaryotic chromosomes Have tandem repeats usually of T1-4G1-4 - With A-C on the opposing strand Can be tens of thousands of base pairs long in mammals TG strand is longer than its complement, leaves a 3’-overhang of several hundred bases THE MECHANISM OF TELOMERASE Telomerase has an RNA (~ 150 Nts) with AxCy repeat to serve as template for synthesis of the TxGy strand of the telomere Telomerase binds to the 3’-end of the chromosome and hangs off so that the RNA template extends beyond it Telomerase extends the 3’-end of chromosomal DNA (the primer), using the RNA of the enzyme as the template (it is a reverse transcriptase) The gap on the bottom strand is filled in by DNA polymerases THE MECHANISM OF TELOMERASE Telomere synthesis and structure: § The internal template RNA of telomerase binds to and base pairs with the TG primer (TxGy) of DNA. § Telomerase adds more T and G residues to the TG primer, then repositions the internal template RNA to allow the addition of more T and G residues that generate the TG strand of the telomere. § The complementary strand is synthesized by cellular DNA polymerases after priming by an RNA primase. SOME VIRAL RNAs ARE REPLICATED BY RNA-DEPENDENT RNA POLYMERASE Some phages (f2 etc.) and viruses (flu, Covid19 etc.) have RNA genomes. These ssRNA genomes, which also functions as mRNAs (RNA+), are replicated by the virus encoded RNA-dependent RNA polymerase (or RNA replicase) RNA replicase functions as any DNA-dependent RNA polymerase – – – – New RNA strand synthesis proceeds in the 5’ à 3’ direction However, RNA is the template (will not function with ssDNA) No proofreading, rate of error similar to that of RNA Pol RNA replicases are specific for their own RNA SUMMARY In this lecture, we learned: RNA-dependent synthesis of RNA and DNA Function of reverse transcriptase and telomerase