Download 9/17/08 Transcript I

Fundamentals Transcript
9-17-2008
10 - 11 (Ryan)
Please follow slides carefully, he jumped around quite a bit. I will only be numbering the slides he talked
on, not the ones he skipped or left out of the lecture.
Gene Expression - Prokaryotic Gene Transcription - Part I - slide 1
Modified Central Dogma of Francis Crick (1958) - slide 2
 This is just the modified central dogma of Francis Crick (1958).
 This just shows the overall flow of genetic information. How DNA is transcribed into RNA and RNA
translated into protein.
Prokaryotic Chromosome (E. coli) - slide 3
 Ok, so this is a prokaryote, E. coli.
 This is the bacteria nucleoid.
 The genome of E. coli consists of a singular circular chromosome of about 5 million base pairs.
 The structure is condensed into a tight compound known as the nucleoid.
 The DNA is organized in 50 -100 loops.
 The question that we're are going to talk about this hour, is how does E. coli determine amongst the
1000 or so genes in the chromosome, which ones to transcribe at any particular time, how does it
identify where the genes are, and how does it regulate which genes are expressed at any one time.
Additional Forms of RNA - slide 4
 Ok, so now we're going to be talking about different forms of RNA. There are 3 different forms that
are found in all cells.
 Those are: messenger RNA (mRNA), ribosomal RNA (rRNA), and the third one is transfer RNA
(tRNA).
 All 3 RNAs are produced by a single enzyme and that is a DNA dependent RNA polymerase.
 In eukaryotes the 3 RNAs are produced by 3 different enzymes, DNA dependent RNA polymerases I,
II, and III. We'll be talking more about that tomorrow.
RNA Quantity and Stability - slide 5
 rRNA and tRNA are fairly stable. They are not degraded rapidly. They are extensively processed.
 In rapidly growing E. coli, about 80% of the RNA is rRNA and about 15% is tRNA.
 The ribosome number [rRNA] is proportional to growth rate. So, the faster the cell is growing the
more rRNA.
 Now the mRNA, which is actually what is translated, is only about 2 to 5% of total RNA.
 mRNA its fairly unstable with a half life (t1/2) of about 2-3 minutes. This rapid turnover allows
regulation at the level of mRNA synthesis or transcription.
mRNA Degradation by RNases - slide 6
 Ok so once an mRNA is produced it can be degraded by RNases.
 There are exonucleases that can chew up the RNA at the 3' region, going from the 3' to the 5'
direction.
 And, there are endonucleases, that can cut internally on the mRNA molecule.
Transcription: The Players - slide 7
 Transcription needs several components.
 First is, ribonucleotides (NTPs) as compared to dNTPs which are necessary for replication. We also
need a template - the DNA template, which is going to be transcribed into RNA, we need an enzyme,
the DNA dependent RNA polymerase, and we need protein transcription factors.
Transcription: DNA to mRNA - slide 8
 Ok, so this is a figure from your book.
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As by convention, when we show a gene or DNA, we will show a double stranded DNA, with the top
strand will read 5' to 3' orientation and the lower or bottom strand will go from 3' to 5'.
By convention the lower strand is called the template strand, the upper is the non-template strand.
And this bottom strand, the template strand, is what the RNA polymerase is reading.
The transcript that is made is complementary to this template strand.
So you can see this first T A C is now a A U G in RNA.
You can tell that every adenine is transcribed to a uracil in RNA, and we'll talk about that later.
In this RNA transcript that is growing 5' to 3' direction, is then translated into protein. These are in
triplets.
So this template strand is read in a 5' to 3' direction and makes the 5' to 3' growing mRNA.
Major bases found in DNA and RNA - slide 9
 The major bases found in DNA are adenine, cytosine, guanine, and thymine (A, C, G, and T).
 And in RNA they are adenine, cytosine, guanine, but now the thymine is replaced with uracil (U).
 In DNA you have the thymine-adenine base pairs and in RNA we now have the uracil-adenine base
pairs.
 And similar to A-T base pair, the U-A hydrogen bonds with 2 hydrogen bonds.
DNA Dependent RNA Polymerase: Catalysis Reaction - slide 10
 Now the actual catalysis reaction of the DNA dependent RNA polymerase.
 You have the template strand of DNA going from 3' to 5' direction and the growing strand of RNA is
synthesized 5' to 3'. So the direction of synthesis is 5' to 3'.
 There is no primer required, but you do need this template.
 The incoming nucleotide, there is an attack of this 3' OH to the alpha phosphate liberating a
pyrophosphate. This pyrophosphate is then cleaved by pyrophosphatase into 2 phosphates, essentially
making this a one way reaction. You get a new formation of a new phosphodiester bond and the strand
is now grown by 1.
Chromosome is divided into genes, which encode RNA and protein products - slide 11
 Ok now, the chromosomes are divided into genes, and we'll talk about how you'll identify these genes.
 These genes code the RNA and protein products.
 Again, so this lower strand is transcribed into the growing RNA from 5' to 3'.
 These bases in the mRNA are complementary to those on the lower strand.
 There are some chemical differences:
 We have a ribose instead of a deoxyribose, uracil is used in RNA versus thymine. This growing
transcript is single stranded, and again it is complementary to the lower strand of DNA.
Naming the DNA Strands of a Gene - slide 12
 Ok, so again by convention, the upper strand is 5' to 3', it is known as the non-template or nontranscribed strand.
 And the bottom strand is the template or transcribed DNA. This is complementary to the transcript
made.
Both DNA Strands Encode Genes And Can Be Transcribed - slide 13
 Genes can occur in either orientation in the DNA molecule.
 So here we have gene #1 drawn from 5' to 3', but its just as likely that another gene can be transcribed
in this direction.
 The promoter is the recognition sequence that helps the RNA polymerase find the gene and know
where to start transcribing.
 Promoters are found upstream from a gene, or 5' of this gene or the other gene the other direction.
 So either DNA genes can be in either orientation.
Prokaryotic RNA polymerase - slide 14
 Ok, so Prokaryotic RNA polymerases transcribe all the major classes of RNA as mentioned earlier.
 It's a multisubunit protein.
 The holoenzyme is composed of 2 alpha subunits, a beta subunit, beta prime subunit, and a sigma
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subunit. This catalyzes the initiation of RNA synthesis, specifically at a promoter.
Once that transcription has been initiated, then the core enzyme catalyzes the elongation of the RNA
chain.
The sigma factor is released and the core enzyme composed of 2 alpha subunits, beta and beta prime
subunits, elongates the transcript.
Transcription in Prokaryotes - slide 15
 In E. coli, the RNA polymerase is a large, multi protein 465 kD with 2 alphas, 1 beta, 1 beta prime and
1 sigma subunit.
 The alpha subunits are essential for assembly and for activation of enzyme by regulatory proteins, and
we'll describe that a little later.
 The beta subunits binds the NTPs, interacts with the sigma factor, and forms a catalytic site with beta
prime.
 So, beta prime subunit binds with DNA and forms a catalytic site with beta.
 The sigma factor recognizes the promoter sequences on DNA, aids in the melting of the double
stranded DNA (dsDNA) by binding the non-template strand.
Prokaryotic Transcription Cycle - slide 16
 The transcription cycle is composed of initiation.
 In initiation the holoenzyme must recognize the promoter, has to unwind or open the promoter, and
starts to form the growing transcript, initially its just small 7 - 12 nucleotides. And you need to have
the sigma factor to recognize the promoter.
 Once you have 7-12 nucleotides formed the sigma factor is released and elongation with the core
enzyme can take place.
 And finally at the end of the gene, end of the transcript, it is terminated.
 The polymerase dissociates from the template DNA and releases the new RNA molecule. And we will
talk about 2 different ways of termination. You can have a Rho factor dependent or independent
termination.
Binding of RNA Polymerase to Template DNA - slide 17
 So polymerase can bind non-specifically to DNA with fairly low affinities and it migrates looking for
these promoters.
 And it is the sigma factor on this holoenzyme that does this recognition of the promoter sequence.
 Once the holoenzyme finds a promoter it forms this "closed promoter complex" and then it opens the
duplex DNA to form the "open promoter complex."
RNA Polymerase Binding to DNA - Promoter Search - slide 18
 So this non-specific binding to DNA, you can think of this polymerase as sliding up and down the
DNA, fairly non-specifically. It has a association constant of about 10^7/M.
 Once it finds a promoter, it has a much more specific, tight interaction, increasing to about 10^14/M.
And this actually depends on the individual promoter, some bind very tightly, so there's a range.
 Ok, so in E. coli there's about 1000 promoters, 1000 molecules sigma factors, and about 3000
molecules of the RNA polymerase core enzyme. There is an unlimited number of nonspecific DNA
sites, so out of those 1000 promoters, that holoenzyme has to be able to find those promoters.
 Question is asked about K-association and K-dissociation. Answer to question = that's just measuring
the strength of binding of polymerase to the promoter, to the DNA in the nonspecific case. So its the
two of them together vs. the two of them separated and the ratio is the K-association and the Kdissociation is the inverse.
Properties of Promoters - slide 19
 Ok so what's a promoter.?
 A promoter in prokaryotes typically consists of a 40 base pair region on the 5' side of the transcription
start site.
 There are typically 2 consensus sequence elements:
 There's the "-35 region" with a consensus TTGACA
 And what's know as the Pribnow box near -10 region, with a consensus of TATAAT, or some people
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also call it the TATA box.
Ok, so -10 and -35.
These consensus sequences shown here are for the sigma 70 factor, which is the most common sigma
factor in E. coli.
Different sigma factors will have different consensus sequences at the -10 and -35 regions. This is one
way the cell can choose and regulate which genes it expresses at any one time.
Prokaryotic Promoters - slide 20
 So, this is a figure from your book that shows different operons in E. coli.
 Different genes are being arranged 5' to 3'.
 It shows the transcription start site, referred to as +1; and the -10 Pribnow box and the -35 region.
 So, -10 refers to 10 bases to the 5' side to the transcription start site. And -35, average about 35 bases
to the 5' transcription start site.
 You'll notice special things about these consensus sequences. The initiation start site is 93% of the
time is a purine.
 These consensus sequences are made by aligning a bunch of genes together and looking to see if there
are any similar sequences that line up in these promoters.
 The Pribnow box, TATAAT, this A position, 95% of the time its an adenine and this final T 96% of
the time its a T, but certainly not a 100%, you will see differences in these genes.
 And again, in the -35, this is just an average consensus of a bunch of genes.
 Again, this is for the 70 sigma factor.
Consensus sigma Factor Promoters - slide 21
 Now depending on other sigma factors have other consensus sequences that they associate with.
 You can see in sigma 32 E. coli it has a different -10 and -35 regions.
 And in B. subtilis, it has several different consensus sequences.
 So again, this allows organisms to turn on different sets of genes under different environmental
conditions.
Stages of Transcription - slide 22
 Ok so lets go through the stages of transcription.
 Here we have again the holoenzyme that is sliding up and down the DNA has to find the promoters
and form a "closed promoter complex," unwind the DNA forming the "open promoter complex,"
initiate polymerization, so the formation of new mRNA transcript, sigma factor drops off and you get
chain elongation, and then finally termination of the transcript.
Transcriptional Events - slide 23
 So, schematically we have the holoenzyme, here's the core polymerase, and the sigma factor shown in
purple, sliding up and down the DNA, it binds it -10 and -35 region that this particular sigma likes to
react with. It stops and forms its closed promoter complex, it then forms its transcription bubble. So
this is now the open promoter complex, begins to have these little aborted transcripts of
ribonucleotides are formed, once those little transcripts get to 7-12 nucleotides in length the sigma
factor dissociates and the core enzyme continues to travel down the DNA, with this open bubble
extending this transcript.
 You can see this transcript growing 5' to 3'.
 This will continue down until it finds a termination sequence, then the transcript DNA in polymerase
will dissociate from one another.
Initiation of Polymerization - slide 24
 The initiation of RNA polymerase has 2 binding sites for NTPs.
 The initiation site prefers to bind ATP and GTP, most RNAs begin with a purine at the 5' end. the
initiation site prefers a purine nucleotide.
 Elongation site binds the second incoming NTP.
 The 3' -OH of the first attacks the alpha-P of the second to form a new phophodiester bond, eliminating
pyrophosphate.
 Once you have about 7-12, oligonucleotide have been made, the sigma subunit dissociates, completing
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initiation.
Note that certain drugs, such as rifamycin, will specifically bind and block prokaryotic gene
transcription. It binds to the beta subunit of RNA polymerase and blocks the first phosphodiester
bond. This drug is specific for prokaryotic RNA polymerase.
Events at initiation of transcription - slide 25
 Now, just schematically once again, the have the holoenzyme and DNA, it slides along finds a
promoter forming a closed promoter complex, then forms the open promoter complex and will unwind
the DNA a little bit forming the open promoter complex. You will have this small abortive initiation
until you get your 7-12 nucleotides. Then the sigma factor will then dissociate and the polymerase will
have structural, and will slide down the DNA transcribing the remainder of the gene.
Chain Elongation - slide 26
 The chain elongation, involves the core polymerase with no sigma factor involved.
 Polymerase is very accurate, only about 1 error in 10,000 bases. That may seem high, but its not
because many transcripts are made from each individual gene, so these errors can occur in many
different places and essentially have no effect.
 The elongation rate is about 20-50 bases per second, this slows down in the G:C rich regions primarily
because it is harder to open up the transcription bubble, because it is 3 hydrogen bonds holding them
together instead of 2. When going through A:T rich regions, it will flow much faster.
 And there are also topoisomerases that precede and follow the polymerase to relieve supercoiling. As
that bubble opens up you get positive supercoiling formed in the DNA in the transcription bubble you
get negative supercoiling. So you have topoisomerases preceding and following to help wind and seal
it back up.
The Elongation Complex - slide 27
 The RNA polymerase (RNAP) core enzyme covers about 60 base pairs of DNA, with about 17 base
pairs unwound, this is the transcription bubble.
 That bubble must contact the active site for you to get polymerization of the growing transcript.
 At the beginning of the bubble, the DNA is unwound, implicating a helicase activity.
 At the end of the bubble, the DNA is rewound.
Supercoiling Versus Transcription - slide 28
 This is a figure from your book.
 Here you have RNA polymerase on the DNA template with the transcription bubble. If this is the way
transcription occurred you would have a mess trying to get the RNA polymerase away.
 And below, you have the topoisomerases preceding and following the transcription bubble, unwinding
the DNA helping to remove supercoiling.
 You see the first topoisomerase relieving negative supercoiling (trailing transcription bubble), so the
RNA polymerase can be removed as transcription proceeds, and the second topoisomerase relieving
positive supercoiling from the RNA polymerase.
Inhibitors of Transcription - slide 29
 Shown here are 2 inhibitors of transcription.
 Actinomycin D, this works by intercalating, between G:C base pairs. Its not specific, it works in both
pro- and eukaryotes.
 Right side is Rifampicin, works only in prokaryotes. It blocks the formation that phosphodiester bond,
by binding in the active site of the beta subunit of RNA polymerase.
Transcription Termination - slide 30
 There are 2 different mechanisms for transcription termination for prokaryotes.
 The 1st is Rho factor dependent mechanism. It is an ATP-dependent helicase. It moves along RNA
transcript after binding it and finds the bubble, unwinds it away from the DNA and releases the RNA
chain from the DNA template.
 The 2nd method is factor independent. It requires specific sequences in the DNA transcript. These are
composed of inverted repeats, rich in G:C, which forms a stem-loop in RNA transcript.
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If you have a sequence, a palindrome, if you transcribed a region like this and you have this sequence
that can base pair with that sequence, they can come together through the hydrogen bonding of base
pairs. Usually in between there the is a little loop, and DNA doesn't participate in = stem-loop.
In the factor independent terminators, this is also called intrinsic termination. Normally, G:C rich
stem-loop, followed by A's that codes for U's in transcript. Then U:A base pair is less stable than G:C
and U:A's can come apart. And by forming that stem-loop you are terminating transcription by pulling
out the RNA.
Transcription Termination Sequence Dependent/Factor Independent (Intrinsic Termination) - slide
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 So, it's easier with a picture.
 Here you have the DNA and this is the inverted repeats in the DNA that will form the stem. And here's
the little loop, the sequence in between. So as you're transcribing the DNA here, you form the stemloop, and that's followed by U's. And so you form as this new transcript is formed, when this sequence
is transcribed you can hydrogen bond with the complementary sequence on the other side forming this
stem, and the whole transcript is released out of the bubble, by the formation of this stem.
 If there are a bunch of G, C's here then it will not terminate.
 So this intrinsic needs the U's.
Factor-independent transcription termination of E. coli - slide 32
 So once again, here we have the DNA, here's the transcription bubble and transcript coming out of the
DNA, and here in yellow are the regions that are complementary that will form the stem-loop. Then
this run of U's formation, the stem loop is able to pull the U's out of the transcription bubble.
Transcription Termination: Rho-factor Dependent - slide 33
 Here is the 2nd method, that requires the factor Rho.
 Once again, we have the duplex DNA, transcription bubble, RNA polymerase. As transcript comes
from bubble, a Rho looks for a binding site, usually a C rich region on the transcript, it has to be free of
ribosomes. If it can find a binding site in this new transcript it will bind Rho x, again it is a ATPdependent helicase. By hydrolyzing ATP for energy it can run up the message and unwind the
transcript from the DNA in the transcription bubble.
 The figure also shows a red region in the transcription bubble the RNA polymerase is stalled (G:C rich
region) and this allows Rho to bind and catch up with the bubble and unwind the transcript from the
bubble.
 This is factor dependent, Rho termination.
Now moving to Gene Expression Part 2 - Regulation of Prokaryotic Gene Transcription
Regulation occurs at every level - slide 34
 Regulation of prokaryotic gene transcription occurs at many levels. During the actual RNA synthesis,
synthesizing the transcript, the main way is regulated, but is also regulated by RNA stability of the
transcript, processing, localization, during translation, and post-translation regulated.
 Lots of ways the prokaryote can regulate the products being produced.
Regulation of Prokaryotic Gene Transcription - Introduction - slide 35
 We will concentrate on the main way to regulate. This requires a DNA protein and protein-protein
interactions.
 Regulation also occurs by organizing genes into operons, we'll specifically talk about the lac and
tryptophan operons.
 We'll talk about positive and negative control of operons, and attenuation control of transcript.
Transcription Terminology - slide 36
 Promoter = a DNA site recognized by RNA polymerase for specific transcriptional initiation
 Terminator = the region of DNA containing signals for termination of transcription
 Structural gene = DNA that encodes a protein (or RNA product?)
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Cistron = another name for a gene, mRNA specified by the structural gene
Coding region = structural gene or cistron, this is the actual region that is transcribed
Open reading frame (ORF) = coding region, has no stop codons. You can have multiple ORF, but only
a single promoter, single start site.
Operon = promoter + the number of genes + the terminator. The operator is a part of the operon,
which helps control the transcription of the promoter of that operon.
General Rules for DNA Binding Proteins - slide 37
 Proteins that are interacting with DNA. A lot of the proteins that bind with DNA are dimers.
 The 3D structure of regulatory proteins, most of them are homodimers.
 DNA sequence recognized by homodimers are typically palindronic, or inverted repeats, and they have
dyad symmetry; here's an example down here. Where this sequence reading left to right is similar to
this sequence reading left to right. This is a palindronic sequence or inverted repeat.
 Usually if you have a homodimer, one of the proteins of that homodimer will recognize this sequence,
and the other one will recognize this sequence. By forming a dimer you can increase the specificity of
DNA protein binding.
 Now you need this inverted repeat sequence to occur in order for that homodimer to interact with the
DNA.
Transcription factors - slide 38
 So the proteins that bind to DNA that effect transcription are called transcription factors.
 DNA binding proteins that decrease the efficiency of transcription at the promoter are called
repressors, and those that increase the efficiency of transcription at the promoter are called activators.
 Typically these proteins will have a couple domains, one that interacts with DNA. Usually this is in
the major groove of the DNA double helix. They'll also have protein-protein interaction domain that
enables to dimerize forming homo and heterodimers.
 The binding site shown here is an inverted repeat, with dyad symmetry, allowing the same protein
forming a homodimer for both sequences.
Transcription is the primary site... - slide 39
 Transcription is the primary site of control in prokaryotes for expression of genes.
 Promoters drive the expression of those genes.
 In Prokaryotic genes, they can be arranged in operons. Where the expression of multiple genes are
controlled by a single promoter and occurring in a single polycistronic transcript.
 Here's an example of a monocistronic operon has a promoter, a single structural gene, and a terminator
downstream.
 Versus a polycistronic operon, with single promoter making a single polycistronic transcript that will
encode for 3 different structural proteins. By organizing genes in operons like this, you can control
expression of all 3 genes with a single promoter.
Transcription Regulation in Prokaryotes - slide 40
 Genes encoding for enzymes of metabolic pathways are grouped in clusters on the chromosome called
operons.
 This allows coordinated regulation and gene expression.
 A regulatory sequence adjacent to such a unit (operon) determines whether it is transcribed. This is the
operator.
 Regulatory proteins interact with operators to control positively or negatively transcription of the
genes.
Coordinate Regulation - slide 41
 Organisms can coordinate regulation.
 An operon is a set of genes that are transcribed from the same promoter and controlled by the same
operator site and regulatory proteins.
 A regulon is a set of genes or operons expressed from separate promoter sites, but controlled by the
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same regulatory molecule. So you can globally regulate expression of many genes at once.
So E. coli when it runs out of glucose as a carbon source, it needs to come up with another game plan.
So it may turn on what ever carbon substrate that is available and will turn on those operons
specifically. And can do this globally.
Global Regulation Via Sigma Factors - slide 42
 Here is an example of global regulation via sigma subunits.
 In this table, it is a list of sigma subunits, and the relative amounts found in a cell, and the number of
genes they can coordinately regulate.
 Sigma factors recognizing this specific promoter sequence, different sigma factors recognize different
promoters, so depending on which sigma factor you produce you can turn on different sets of genes.
 So this is showing 2 growth conditions, a growing phase of a culture vs. a stationary phase. Once you
have some many E. coli and start running out of nutrients they stop exponential growth phase and go
into this stationary phase. When that happens you turn on the sigma factor RPoS, shown in blue, so
you start making this sigma factor and this turns on the stationary phase genes. so you get a whole set
of different genes expressed as E. coli runs out of glucose for substrate and goes into stationary phase,
slow to no growth.
BREAK BEGINS!!!