340 - 05 -- processing
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• 5’ untranslated region of mRNA can
switch between 2 conformations
• Antiterminator: allows transcription
to continue
• Terminator: stops transcription
• Switch is dependent on binding of a
small molecule to 5’ untranslated
region
Insect metamorphosis: regulated by ecdysone
Amphibian metamorphosis: regulated by thyroid hormone
Male vs. female development: regulated by testosterone and estrogens
All of these hormones have receptors that bind to DNA (zinc-finger proteins)
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Heritable changes not due to changes in DNA sequence itself (epi = besides or in addition to)
Chemical modification of bases• Cytosine methylation• After incorporation, DNA methylase adds methyl group
to 5 carbon• Methylation occurs more often if C is next to G• During replication, if both parent strands are
methylated, newly made strands will then be methylated by methylase
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Lots of methylation – lower transcription• X chromosome inactivation in female mammals
Accompanying video fair game
Transcriptional cosuppression• Extra copies of genes induce higher levels of
methylation (in all copies, not just the extra ones)
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Genetic imprinting• In germ line only (gamete forming cells)
• A few hundred genes are methylated
• Different methylation in females vs. males
• Imprinted genes are inactive during
embryogenesis (early development)
• Imprinting erased in germ cell development, re-
established later
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Insulator DNA sequences• Bind specific proteins
• Prevent enhancers from activating some
promoters
• Prevent chromatin condensation from interfering
with transcription
Acetylate nucleosomes on either side of actively
transcribed gene
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RNA selection: not all RNA is transported out of nucleus
RNA splicing• Translation doesn’t start at 5’ end of primary transcript
• Primary transcript includes: 5’ untranslated region
3’ untranslated region
Introns and exons (non-coding and coding regions)
Eukaryotes - must be processed prior to translation
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Three changes to primary transcripts
Two are “End modifications”• 1) 5’ “cap”
7- methylguanosine added to 5’ end
• 2) “Poly A tail”
Adenines added at 3’ end
The third is internal• 3) Splicing
Removes specific sequences from central part of
primary transcript
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Coding regions are interrupted by noncoding segments - “split genes”
Coding regions - exons (ex for expressed)Noncoding regions - introns (intervening
sequences)Primary transcript contains exons and
introns RNA processing removes introns and
pastes exons together
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Number & size vary among genes &
species
Most eukaryotic genes have introns
Most of intron sequence may not be
important, but some sequence is important
Splice site sequences are important
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gene feature Median mean
Internal exon 122 bp 145 bp
# of exons 7 8.8
Size of introns 1023 bp 3356 bp
5’ untranslated 240 bp 300 bp
3’ untranslated 400 bp 770 bp
Coding sequence 1101 bp 1341 bp
Amino acids 367 bp 447 bp
Total length 14,000bp 27,000bp
14Don’t need to know this
Some genes have lots of introns and exons:
For example: Tropomyosin:• Structural protein associated with cytoskeleton
• Gene in mammals has 11 exons
Different tissues splice the exons together differently
Not all exons are used in some tissuesSo… resulting proteins also differ among
tissues
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Different splicing results in different mRNA
Different mRNA’s produce different (although related) proteins
Proteins can vary in function
Common process (up to 75% of all genes may have alternative splicing)
Produce more proteins than you have genes
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mRNA stability: Longevity of mRNA determines how many proteins can be produced
• Deadenylation-dependent: enzymes trim poly-A tail, when poly-A tail is short enough (25-60 bases), mRNA can be “uncapped” which stops translation and leads to mRNA degradation
• Deadenylation-independent: decapping or endonuclease (enzyme cuts up mRNA) Common in mRNA’s with early stop codons, or unspliced
introns
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RNA interference (RNAi): post-
transcriptional silencing mechanism• Relies on production of double-stranded RNA’s
RNAi sequences complementary to short region of
mRNA
Double stranded RNA is recognized by cell and
enzymatically digested
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Germ line DNA has lots of unique sequences for variable regions of antibodies
Germ line DNA has lots of unique sequences for joining regions of antibodies
Splicing combines variable and joining regions randomly
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Results in lots of different combinations
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