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Exam 1Mean = 66
Median = 66Range (26 94)
A > 84 (21)B 74 to 82 (34)C 64 to 72 (45)D 50 to 62 (56)F < 48 (25)
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0102030405060
A B C D F
Exam 1 Grade Distribution
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Chapter 7From DNA to Protein: How Cells
Read the Genome
EssentialCell Biology
Third Edition
Copyright Garland Science 2010
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Francis Harry C. Crick James Dewey Watson
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The Central Dogma of Molecular Biology
Occurs in all cells frombacteria to humans.
One of the definingcharacteristics of livingcells.
Allows massiveamplification of signals
from a single gene.
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The efficiency of gene expression
is quite variable
Translation efficiency, as well asRNA and protein stability vary
greatly among genes
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Structure of RNA
Differs from DNA in 2 significant ways:1. ribonucleotides vs deoxyribnucleotides2. uracil vs thymine
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RNA is typically single stranded
Because RNA is single stranded, intramolecular base pairingcan occur, resulting in elaborate secondary structure This gives rise to diverse functionality (e.g., ribozymes,
riboswitches, tRNA, rRNA, )
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RNA serves many functions
final product = RNA molecules
Gene expression refers to the biosynthesis of eitherDNA-encoded protein, or non-coding RNA
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From an evolutionary
perspective, RNA may have been
the original, self-replicating
biopolymer
Ribozymes may have developed
the ability to direct protein
synthesis
DNA is probably a relative
newcomer, usurping RNAs role
in information storage
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Transcription is the DNA-directed biosynthesis of a
single, complementary RNA molecule
RNA is much shorter than DNA. RNA polymerase carries out transcription. RNA polymerase does not need a primer. Many RNA polymerases can transcribe a
single gene at the same time.
Transcription does havesimilarities to replication.
As for DNA, RNA issynthesized in the 5to 3
direction.
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Figure 7-7 Essential Cell Biology ( Garland Science 2010)
*RNA polymerase = 10 subunit protein complex
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Eukaryotic transcription differs from bacterial
transcription in a few ways
1. Bacteria have only 1 RNA pol. Eukaryotes have 3.
2. Eukaryotic RNA polymerases require a bunch of accessory proteinscalled the general transcription factors (GTFs) to initiate
transcription.3. Control mechanisms are more complex in eukaryotes in part becausegenes are much further apart. This allows more sophisticated gene
regulation.4. Eukaryotic transcription has to deal with more compact chromatin
structure.
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Bacterialpromotersand terminatorshave specific
sequences recognized by RNA polymerase
The promoter orients RNA pol and tells it where to start and whichway to go.
All bacterial genes have promoter and terminator sequences similar tothose shown below.
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The General Transcription Factors
Assemble on the promoter. Position RNA polymerase. Open DNA. Launch the RNA polymerase.
typically ~25 bp upstream
of start site; mostpromoters have this
transcription initiation
complex
Both opens DNA and
phosphorylates and
releases RNA pol from
initiation complex
TBP is a
subunit ofTFIID that
distorts
DNA,
forming
landmark
All components are then laterrecycled to be used again and
again
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Figure 7-13 Essential Cell Biology ( Garland Science 2010)
TATA Box Binding Protein distorts the double helix*
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Figure 7-12 (part 1 of 2) Essential Cell Biology ( Garland Science 2010)
~ 25 bp upstream
from transcription
start siteTBP distorts DNA
TFIIB provides scaffold
TFIIH separates strands
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Figure 7-12 (part 2 of 2) Essential Cell Biology ( Garland Science 2010)
TFIIF phosphorylates tail
domain of RNA pol II
. launches polymerase
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Control of transcription initiation
is the most common wayorganisms control gene
expression.
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In eukaryotes, transcriptionoccurs in nucleus, translation
occurs in cytoplasm. The
exportof RNA occurs via
nuclear pore complexes in the
nuclear envelope.Prior to nuclear export,RNA
processingoccurs as the RNA
molecule is being synthesized.
Eukaryotic RNAs must be processed and
exported to cytoplasm
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Occurs as RNA is beingmade. Processing machinery is
recruited to the
phosphorylated tail
domain of the eukaryotic
RNA polymerase. Different types of processing
occurs depending of what
type of RNA is being made.
RNA processing *
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Addition of 5
-caps and 3
-polyadenylation tails (poly-A tails)
(also intron splicing) . 5-caps and poly-A tails:
1. Increase stability.2. Identifies the molecule as mRNA.3. Marks the mRNA as being
complete.
Eukaryotic mRNA processing
Usually gets trimmed back firstbefore few hundred Aadded.
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Eukaryotic genes are often interrupted by noncodingsequences (introns). Need to remove/splice these introns out to
get finished/meaningful message. Exons-expressed sequences Introns-intervening/nonexpressed sequences
Introns in eukaryotic mRNA are
removed by RNA splicing
Splicing can happen in prokaryotes, but rarely does.
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Occurs during transcription after 5-capping. Can occur before during or after
addition of poly-A tail. Involves cutting out introns and stitching
exons back together.
Introns are removed by RNA splicing
Unlike exons, most of intron sequence appearsto be unimportant. There are a few shortsequences at or near each intron end that act as
cues for removal.
carried our primarily by catalytic RNA
molecules (small nuclear RNAs; snRNAs)
coupled to a few proteins to form small nuclear
ribonucleoprotein particles (snRNPs)forming
the core of the Spliceosome.
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This process carried our primarily by catalytic RNA
molecules (small nuclear RNAs; snRNAs) coupled to a fewproteins to form small nuclear ribonucleoprotein particles
(snRNPs)forming the core of the Spliceosome.
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Figure 7-20 Essential Cell Biology ( Garland Science 2010)
snRNPs bind to specific
sequences at both ends of
the intron.The 2hydroxyl of a
conserved Aattacks 5
splice site forming lariat3hydroxyl of first exon
attacks 3splice site,
knitting exons togetherLariat is degraded
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Introns are removed by RNA splicing
Alternative splicing leads to greater protein diversity fromsingle gene.
~60% of human genes undergo alternative splicing. Could have helped speed evolution of eukaryotes
(e.g., domain swapping ).
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TheNuclear Pore Complex recognizes and
exports only completed mRNAs
Eventually all mRNA molecules are degraded into nucleotides viaRNases. Sequences in the 3
-UTR help determine stability.
RNA binding proteins interact with and signal properly mademRNAs (cap binding complex, exon junction complex, polyA BP).
RNAs that dont pass QC are degraded and recycled.
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mRNA processing summary
Chicken & Egg questionTranscript lifetimes vary(~ 3 min for procaryotes,
~ 30 min for eucaryotes)
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Translation is the RNA-directed
biosynthesis of proteins
In order to unambiguously specify 20 amino acids using a code
composed 4 letters(bases), need at least 3 letters = codonThere are 4
3
= 64 permutations = unique codons
Thus, the genetic code is redundant some AAs specified by more
than one codon.Punctuation marks = AUG (start; also Met); UAA, UAG, UGA = stop
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RNA can be read in any of 3 open
reading frames
Appropriate ORF is signaled by the translation start site.
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Codons specify binding sites for tRNAs
tRNA = transfer RNA Only 31 different tRNAs for 64 codons. Some tRNAs recognize > 1 codon (wobble
phenomenon).
D = dihydrouridine; != pseudouridine
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Correct chargingof tRNAs requires enzymes
called aminoacyl-tRNA synthetases
Each AA is chargedto one or more UNIQUE t-RNAs.
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Ribosomes are enormous protein-
manufacturing machines
Ribosomes are made of > 80 different proteins (ribosomalproteins) and 4 RNA molecules (rRNAs).
Composed of 1 largeand 1 smallsubunit. Highly conserved.
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Ribosomes are elaborate ribozymes
matches tRNAs
to mRNA codonscatalyzes formation
of the peptide bonds
Subunits separate after protein
synthesis is completed
~ 2:1 RNA:Protein
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Figure 7-32 Essential Cell Biology ( Garland Science 2010)
Three t-RNA binding sites on ribosome
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Figure 7-33 (part 2 of 5) Essential Cell Biology ( Garland Science 2010)
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Figure 7-33 (part 3 of 5)Essential Cell Biology ( Garland Science 2010)
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Figure 7-33 (part 4 of 5) Essential Cell Biology ( Garland Science 2010)
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Codons within the mRNA signal
where to start and stop translation
Translation begins withAUG start codon. Need special initiator tRNA and translation
initiation factors. Special initiator tRNA is used at the start codon
that always carries methionine. Only tRNA that
can bind straight to the P site. This methionine is usually removed later by a specific
protease.
Bacteria use a Shine-Delgarno sequence (~6 bpslong) upstream of the AUG to cue the start of
translation, while eukaryotes use a Kozak
sequences.
cap recognized
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+EF-Tu
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Prokaryotic mRNA is usually polycistronic
Prokaryotic ribosomes can recognize and bind near start codonsin the middle of an mRNA molecule.
Several related proteins are synthesized simultaneously from asingle mRNA
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Translation is terminated at stop codons
UAA, UAG and UGA They signal the
binding of release
factors. A hydrolysis reaction
then frees the
polypeptide. Nascent proteins aretypically met by
chaperone proteins as
they emerge from the
ribosome
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Proteins are made on polyribosomes (polysomes)
During efficient protein synthesis, anew ribosome hops onto the mRNAright after the preceding ribosome
moves out of the way.
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Posttranslational modifications
After biosynthesis, many proteins undergoposttranslational modificationsthat can
substantially alter protein stability and
function.
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Many antibiotics target prokaryotic translation
Weve been able to borrowmuch of this technology from oureukaryotic cousins, the fungi.
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Proteins can have vastly different life spans
Proteins are degraded byproteases. Many proteins are degraded byproteosomes. Requires ubiquitin tags. However, other mechanisms like autophagyalso occur.
proteosome autophagy
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Gene expression summary *
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