Rea Lec 10 Gene Expression FP

8/13/2019 Rea Lec 10 Gene Expression FP

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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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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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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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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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Three t-RNA binding sites on ribosome


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Figure 7-33 (part 3 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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Rea Lec 10 Gene Expression FP

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