Gene Expression - Biology Department | Western Washington

Report
This Week
• Chapters 9, 10.1 and 10.4 - 10.7 for reference,
– exam material will be on lecture content for the above,
– sample questions, and questions for these chapters will
be posted this afternoon.
• Exam Friday: Assignments in Chapters 6, 11, 8, 9
and 10. All lecture material.
Gene Expression
…the processes by which information contained in
genes and genomes is decoded by cells,
...in order to produce molecules that determine the
phenotypes observed in organisms,
– transcription (post-transcriptional modifications),
– translation (post-translational modifications.
Transcription
...the synthesis of mRNA from a DNA template,
- now it is important to understand when and where,
as well as how.
mRNA Synthesis
• Template (DNA) and Promoter,
• Nucleoside triphosphates (NTPs),
– N: A,U,G,or C,
• Enzymes (RNA polymerases),
• Energy (as in replication, from phosphate bonds).
E. coli RNA Polymerase
RNA Polymerase Scanning
E. coli Promoter Sequences
Promoter Regions
Proximal ( ~ 100 - 200)
Core Promoter ( ~ 0 to ~ -40 bp)
Regulation of Transcription
(Prokaryotes)
• Regulation of gene expression is often at the
transcription level,
– Negative regulation,
• inducible,
• repressible,
– Positive regulation
Gene native state is “on”.
Gene native state is “off”.
Negative Regulation
(Inducible)
Protein!
Protein, or metabolite, etc.
…default state is “on”,
i.e., the gene is
transcribed.
Negative Regulation
(Repressible)
Protein!
Protein, or metabolite, etc.
…default state is “on”,
i.e., the gene is
transcribed.
Positive Regulation
…default state is “off”,
i.e., the gene is not
transcribed.
Prokaryote Review
> 8 sub-units
Basal Factors
TATA-binding protein.
TFIIA binds to the TFIID
complex to block inhibitors
(D-A complex).
TFIIB binds to the D-A complex.
RNA polymerase II
and TFIIF complex
bind.
TFIIE, TFIIH and TH FIIJ added in order.
Eukaryotic Initiation
Promoter Bashing
Eukaryotic Enhancers and Silencers can act at great distances.
Drosophila Guts and Such
dpp locus
...(c) and (d) are ID
enhancer driven,
give rise to fly
appenages.
Terms
• cis-acting elements;
– DNA sequences that
serve as attachments
sites for the DNAbinding proteins that
regulate the initiation
of transcription.
• trans-acting elements;
– the DNA-binding
proteins that regulate
the initiation of
transcription.
Chromatin
Remodeling
Expressed Proteins
Alternate Promoters
(Figure 10.22)
Transcription Unit
…the portion of a gene that specifically codes
for a protein (cistron), plus other mRNA.
5’
3’
transcription unit
RNA polymerase begins transcription here, just
“upstream” of the DNA that codes for the protein.
Untranslated Regions
5’ UTR
3’ UTR
UTRs are transcribed, but not translated.
UTR sequences diverge more rapidly during evolution
than structural regions.
Terminator
…a sequence of nucleotides (AAUAAA in the
transcribed molecule) that specifies the end
of the transcription unit.
Terminator
5’
3’
transcription unit
RNA polymerase begins
transcription here.
RNA Synthesis
…from one strand of
the double helix,
DNA template strand
is read 3’ to 5’,
RNA strand ‘grows
from 5’ to 3’,
Elongation
Nucleotides are added to the 3’ end of the elongating RNA.
mRNA vs. pre-mRNA
• prokaryotic mRNA synthesis described so
far requires little, or no further modification
prior to translation into proteins,
• eukaryotic mRNA requires extensive
modifications.
Post Transcriptional Modification I
5’ UTR
3’ UTR
…modified guanine cap added to the 5’ end.
…lots of
adenines added
to the 3’ end.
Occurs in the nucleus.
Increases stability, may help transport and sorting.
Post Transcriptional Modification II
…intervening sequences (introns), do
not code for proteins.
…code for proteins.
Introns affect expression.
Differential splicing can alter the protein’s function.
Provides “functional cassettes”, for evolutionary mixing and matching.
small nuclear Riboproteins
(snRNPs)
• Introns are spliced
out at structure
called
spliceosomes,
• mRNA remains
relatively stable,
introns are digested
rapidly.
Splicosomes
snRNP = small nuclear RiboNucleoProtein.
Study Figure 9.15 for Wednesday
Intron Excision
Alternate Excision
(example)
Genetic Code
…RNA is an intermediary
in the transfer of
information from DNA
to the synthesis of
protein,
…how is that information
organized?
Code is Linear
The 5’-3’sequence from the DNA template is
equivalent to the mRNA (except for uracils).
DNA code strand...
5’-AAA GGC TGA TCA ATC GAT CGT GAC-3’
DNA template strand...
3’-TTT CCG ACT AGT TAG CTA GCA CTG-5’
RNA strand...
5’-AAA GGC UGA UCA AUC GAU CGU GAC-3’
Information Capacity
• need code for 20 Amino Acids,
• two base-pair code,
2
– 16 different combinations of 4 bases (4 ),
• aa, at, ac, ag, tt, tc, tg, cc, cg, gg, ta, ca, ga, ct, gt, gc,
• three base-pair code,
– 64 combinations of 4 bases (43).
Codons
…a triplet of nucleotide bases that specifies or
encodes the information for a specific
amino acid,
– also need codons to indicate the beginning and
end of the protein to be synthesized.
rII Again
Revertants returned the code to an “in frame” conditions.
Code is Degenerate
20 amino acid codons
+ start and stop codons
20-some required
64 possible
All combinations are used.
Start/Stop Codons
…AUG codes for the ‘start of translation’, a
methionine,
– most proteins thus begin with the amino acid
methionine,
…UAA, UAG and UGA are stop codons,
indicating the C terminus of the protein.
Wobble: the third nucleotide is not necessarily specific.
Central Dogma
DNA
replication
transcription
RNA
translation
Protein
Translation
…the synthesis of a polypeptide. This occurs
on ribosomes using the information encoded
on mRNA,
– tRNA molecules mediate the transfer of
information between mRNA and the growing
polypeptide.
tRNA
amino acid attachment site: each
amino acid has specific tRNA(s),
anti-codon: site of interaction
with the mRNA template.
The ‘Ends’ to the Means
tRNA secondary
structure ‘reveals’ a
three base pair structure
on one end,
Specific anti-codons for specific amino acid designation.
“anti” = complementary
activating enzymes: aminoacyl-tRNA
synthetases, one for each amino acid.
Ribosomes
…a supramolecular complex of rRNA and
proteins, approximately 18 - 22 nm in
diameter,
…the site of protein synthesis,
Ribosome Structure
Structure/Function
E site (exit):
uncharged tRNAs are
discharged from here.
P site (peptidyl): binds the
tRNA that holds the growing
polypeptide.
A site (amino acyl): binds the
tRNA that holds the next
amino acid,
Initiation
small sub-unit and a charged MettRNA (methionine) forms the
initiation complex,
large sub-unit binds the complex,
P site associates with the MettRNA.
1. recognition:
tRNA anti-codon
matches RNA
codon,
Elongation
(3 steps)
(uncharged tRNA exits)
leaves open A
site, cycle
repeats.
3. translocation, tRNAs shift,
mRNA shifts 1 codon,
2. amino acid(s) from P
site tRNA transfered to
the new tRNA, peptide
bond formed,
Peptide Linkage
hydrolysis reaction
Elongation
(3 steps)
Peptide bond
formed.
Termination
stop codon attracts a
protein release factor,
hydrolysis of last
tRNA/amino acid yields
terminal carboxyl group,
ribosome
disassembles.
N-Terminus --> C-Terminus
...polypeptides are synthesized beginning from
the N-terminus (amino terminus) and going to
the C-terminus (carboxy terminus),
…this corresponds to the 5’-3’ DNA Coding
sequence.
Wednesday

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