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Viruses parasites Genome: Double-stranded DNA (circular or linear) Single-stranded DNA, or mRNA (ss or ds) Protein Head: Protect genome while outside cell Tail: Protein for entry into target cell Injects genome Typical viral life cycle Viruses are parasites • Virus genome has promoters Host begins to make viral proteins Many encourage replication Others are viral proteins • Viruses uses host cellular machinery ie- DNA pol, ligase etc. • Viral Genome replicated like host cell does Lysogenic Cycle: To escape host defenses some viruses may integrate into host genome Viroids and Prions: The Simplest Infectious Agents • Viroids are circular RNA molecules that infect plants and disrupt their growth • Prions are slow-acting, virtually indestructible infectious proteins (ex: brain disease mad cow) • Prions propagate by converting normal proteins into the prion version Retroviruses • • • • Retro: RNA DNA Single-stranded RNA genome Many bind and don’t hurt host cell But some do – i.e. HIV •Reverse transcriptase/ integrase A DNA polymerase uses DNA or RNA as template Inserts DNA into host genome Starts at LTR’s like ori Terminal Repeats and unique sequences needed for priming and reverse transcriptase / integrase recognition Reverse Transcriptase (RT) • • • • RT recognizes the DNA sequence “Terminal Repeats” (which are also strong promoters) copies RNA genome into DNA & inserts into genome randomly To complete the life cycle, host RNA pol must transcribe back into RNA RT is 1 of many viral genes Transposable Elements - move from one site to another in a cell’s DNA; they are present in both prokaryotes and eukaryotes Retrotransposons - Defective retroviruses - Present in 1000s of copies - 43% of human genome - requires transcription for movement DNA Transposons - Present in fewer copies – 3% of human genome Retrotransposons • Have lost the genes required to make viruses (e.g. capsid proteins) • Still have reverse transcriptase/integrase gene that increases their copy number in the genome and DNA repeats which they target Needs Transcription to be active • Makes mRNA which is essentially the gutted viral genome • This produces RT • RT is then responsible for integrating new copy into genome elsewhere Active transposition results in duplication as transcription produces multiple copies of the mRNA. DNA transposons • Similar to retrotransposons, but no RNA stage • Terminal repeats • Contains gene for transposase - Recognizes terminal repeats - Cuts and inserts into new location • Mechanism removes transposon but leaves behind a copy of the repeats = “footprints” • Movement requires transcription • Transcription is repressed (silenced) by modifying the structure of chromatin where transposons are located Chromatin structure Almost 2 meters of DNA must be packaged in each human nucleus Chromatin – DNA plus all bound proteins Interphase – time when some genes are transcribe Nucleosome • Most DNA wrapped around octamer of 8 histone proteins • Each histone has a globular domain and histone tail (N-ter) • Histones have many positive charged amino acids (Lys, Arg) that interact with neg DNA Regulation of the degree of coiling is called chromatin remodeling “CG” methylation • • • • • Many eukaryotes (mammals), but not all (yeast, drosophila) ~ 5% of cytosines methylated in human genome. All in the 5’-meCG-3’ sequence context Not all CG’s methylated Both strands methylated Reflects DNA methyltransferase specificity 5’-ACCTCGGCAATCC-3’ 3’-TGGAGCCGTTAGG-5’ DNA methyltransferases (DNMT) are the enzymes responsible for methylating cytosine •De novo methylation – Dnmt3 finds an unmethylated transposons and methylates them. •Once methylation is established, pattern is maintained by Dnmt 1 as cells proliferate C*G C G G C G C C* G C G G C* G C DNA replication Newly synthesized Dnmt1 Hemimethylated C G C G G C*G C C* G C G G C* G C Fullymethylated Dnmt1 Acetylation HAT = Histone acetyltransferase • Adds acetyl groups • Opens DNA HDAC = histone deacetylase • Removes acetyl groups • Coils DNA Less transcriptional activity C*G C G G C*C G Regulation of Eukaryotic Gene Expression Genes are expressed when functional active product (RNA, protein) Regulated at many levels – Chromatin structure – Transcriptional – Post-transcriptional • iRNA - Non-coding RNAs (can degrade other mRNAs) – Translational – Post-translational • Enzymatically – altering activity • Protein degredation Post-transcriptional gene silencing by RISC complex miRNA – micro RNA • Prevents expression • Animals and plants • When RNA forms 22bp – Dicer (ribonuclease) cleaves both strands & exports to cytoplasm – A helicase unwinds & inserts singlestrand into a RISC (RNA-induced silencing complex) (multiple proteins miRNA sequence base pairs to target 1. Causes mRNA degredation 2. Blocks ribosome no translation NEGATIVE Regulation Protein Processing and Degradation • After translation, various types of protein processing, including cleavage and the addition of chemical groups, are subject to control • Proteasomes are giant protein complexes that bind protein molecules and degrade them Ubiquitination Ubiquitin Proteasome Protein to be degraded Ubiquitinated protein Proteasome and ubiquitin to be recycled Protein entering a proteasome Protein fragments (peptides) E3 Ligase Class of enzymes that specify targets through protein-protein interactions