Transcript Bio1A Unit 2-8 Gene Expression Pt 2 Notes File

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
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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)
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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
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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