Mechanisms of Transcription (13) Flashcards
The central dogma of molecular biology
Genetic information is stored in DNA, maintained during replication, and expressed during transcription. Gene expression is conserved across lifeforms as DNA -> RNA -> Protein.
Similarities and differences between DNA replication and DNA transcription
Similarity:
Both involve enzymes that synthesize a new strand of nucleic acid complementary to a DNA template strand (mRNA-strand is similar to daughter strand). This is performed by a polymerase.
Differences:
- The RNA polymerase does not need a primer to start RNA synthesis.
- Newly synthesized RNA is single-stranded and made of ribonucleotides (rNTPs)
- RNA contains uracil (U) instead of thymine (T)
- RNA product does not remain base-paired to the template DNA
- Function: Replication must produce only one stable copy of the entire genome, while transcription makes numerous unstable copies of selected genes.
- Product: Fully replicated genome vs. abundant protein product
How and why is DNA transcription a less accurate process than DNA replication?
Replication: 1 error out of 10 000 000 nucleotides
Transcription: 1 error out of 10 000 nucleotides
During replication, one single stable copy of the DNA will be transmitted to the next generation. If there is one mistake in this copy that isn’t fixed, catastrophic mutations could be transmitted to the next generation and reduce their viability. To avoid mistakes, DNA polymerase has efficient proofreading activity.
Transcription produces thousands of mRNA molecules which are short-lived, meaning that an error will affect only a few mRNAs and then disappear. mRNA with error might not even reach the ribosome. Evolution didn’t think it was necessary with efficient proofreading activity for RNA polymerase.
How do replication and transcription serve different purposes?
Replication must produce only one stable copy of the entire genome, while transcription makes numerous unstable copies of selected genes.
Which regions are transcribed, and how can expression vary between genes and tissues?
The choice of regions to transcribe is not random, as there are specific DNA sequences which trigger transcription initiation. The extent of mRNA synthesis can vary from one gene to another. All genes have the same genome, but different cells (varying tissue, location, environment, time) have very different shapes and functions. This cell differentiation starts at the transcription level, where there are different genes expressed in different cells to create their identity and function. Transcription is a highly regulated process.
What is the number of RNA polymerases in prokaryotes and eukaryotes?
Prokaryotes: 1 RNA polymerase
Eukaryotes: 3 RNA polymerases - RNAP I, RNAP II & RNAP III [+ IV & V in plants]
Who resolved the structure of the RNA polymerase?
Roger D. Kornberg & Patrick Cramer
What characterizes the structure of the RNA polymerases?
Multi-subunit complexes with shared characteristics from bacteria to humans. Two of the subunits are large and conserved and have catalytic activity (active site, synthesis of RNA). In addition, there are several other smaller accessory subunits (less conserved) which are structural or regulatory proteins.
RNA polymerases in prokaryotes and eukaryotes have the same organization and same shape - a crab claw where the active site is located deep within the claw.
The function of the 3 major RNA polymerases in eukaryotes
RNA polymerase II:
- The most studied of all polymerases and the most regulated
- Function: Transcribes protein-coding genes into mRNAs
- Transcribes 20 000 - 25 000 genes in humans
RNA polymerase I:
- Function: Transcribes non-coding ribosomal RNA genes into ribosomal RNAs (rRNAs), the structural component of the ribosome.
- Transcribes 1 gene, making 200 copies
RNA polymerase III:
- Transcribes non-coding transfer RNA genes into transfer RNAs (tRNAs)
- Transcribes 50-100 genes, making 500 copies
The structure of a minimal gene (what 4 overall sequences is it composed of?)
The promoter
- DNA sequence bound by the RNA polymerase (with auxiliary factors) to initiate transcription. Can overlap with +1 site.
The transcription start site (TSS), or +1 of transcription
- Where transcription starts.
The transcribe region, or open reading fram (ORF), or gene body
- The DNA sequence transcribed into RNA by RNA polymerases. Stretches between +1 and TTS.
The transcription termination site (TTS)
- Where transcription ends.
The enzymatic reaction of polymerases - 3 steps, basic principles
RNA opens or “melts” the DNA to create the transcription bubble, which allows for the separation of the 2 strands (template and nontemplate).
RNAPs attach to, travel along and read the template DNA strand 3’ -> 5’ and catalyse the production of a complementary RNA 5’ -> 3’.
The RNA product is the ext same as the nontemplate DNA strand (aka coding strand), except U instead of T.
The polymerization reaction: The assembly line - chemical details, equation
This enzymatic reaction is shared by all prokaryotic and eukaryotic RNA polymerases:
Opening of the DNA -> Base-pairing of rNTPs with template strand -> Formation of new phosphodiester bond
RNA_n + rNTP + (Mg^2+ + template) -> RNA_n+1 + PPi
The phosphodiester bond is formed between the incoming, complementary rNTPs and the template strand. Between the 3’ dioxo-group of the RNA and the 5’ phosphate in the incoming rNTP. The process of adding a new rNTP is called the polymerization reaction. The presence of Mg^2+ catalyzes the formation of the phosphodiesterbond.
Describe the assembly line of polymerases
There are several channels in the polymerase enzyme that allow rNTPs, DNA and RNA in and out.
The DNA enters through the DNA entry channel and deep into the polymerase to reach the active site. rNTPs are brought in through the rNTP entry channel, and the polymerase makes a phosphodiesterbond between the DNA and the incoming rNTPs that are complementary to the template strand. The finished RNA strand exits through the RNA exit channel. The transcription bubble is then closed, and the whole DNA molecule exits via the DNA exit channel.
Channels of the polymerase enzyme
DNA entry channel
rNTP entry channel
RNA exit channel
DNA exit channel
The three steps of transcription
- Initiation
- Elongation
- Termination
Describe the three sub-stages of initiation
- Assembly of transcription machinery (polymerase + auxilary factors) at specific recognized sequences in the promoter -> Closed complex (DNA is closed, duplex)
- Opening of the transcription bubble (RNA melts duplex DNA near TSS) -> Open complex (DNA is opened)
- Polymerase catalyzes phosphodiester linkage of 2 initial rNTPs incorporated, to start the synthesis of the RNA -> Initially transcribed complex
PS! Initiation is the step when most transcriptional regulation occurs
Describe elongation (incl. the range of functions of RNA polymerase)
After forming the initially transcribed complex at the end of initiation, the RNA polymerase will escape the promoter to the gene body. Here it will advance along the transcribed region in 3’ -> 5’ direction while synthesizing RNA along the full length of the gene.
During the travel time along the template DNA, RNA polymerase… :
- Unwinds DNA in front (melting duplex DNA)
- Incorporates rNTPs with phosphodiester bonds
- Dissociates nascent RNA from DNA template
- Rewinds DNA after the reaction (close transcription bubble behind it)
Describe termination
Termination occurs when the RNA polymerase has reached the transcription termination site (TTS) at the end of the gene. Here, RNA polymerase dissociates from both the completed RNA product and the DNA.
Prokaryotes: The structure of bacterial RNA polymerase (subunits)
6 subunits: 2 alpha, 1 beta, 1 beta’ (prime), 1 omega.
RNAP core (a2BB’w)
Prokaryotes: The transcription cycle in bacteria
In vitro, The RNAP core can start transcription from any DNA location, even if there is no gene. On its own, RNA polymerase is completely blindfolded.
In cells, transcription only start at gene promoters. To bring some specificity to the enzyme there is an initiation/auxiliary factor called the sigma factor which associates with the RNAP core to form a complex called the Holoenzyme. Thanks to the sigma factor, the holoenzyme can specifically recognize the promoter.
Which sigma factor is predominant in E. coli?
The sigma factor o^70, which is the most studied and abundant among all the sigma factors found in bacteria.
Prokaryotes: Characteristics of the promoter recognized by o^70-holoenzyme
The two major and most important sequences in the bacterial promotor is the -35 and -10 elements (minus is relative to the +1 of transcription, these sequences are upstream of TSS). These two sequences are separated by 17-19 nucleotides.
There are also additional elements recognized by the o^70 homoenzyme:
- UP-element -> Found at strong promoters of rRNA genes (upstream of -35)
- Extended -10 (lack of -35) -> Found at gal genes
- Discriminator -> Influences strength of holoenzyme-promoter interaction (downstream of -10)
Prokaryotes: Binding of sigma-factor to the promoter (regions and recognition), formation of closed complex
o^70 is divided into four regions, where each region is responsible for the recognition of an element.
Element -35 is recognized by o4, which contains 2 alpha-helices forming helix-turn-helix motif -> Sequence-specific interaction with the major groove of the DNA.
Element -10 is recognized by o2, which contains 1 alpha-helix and is involved in promoter melting at this site.
Extended -10 is recognized by o3, which contains 1 alpha-helix
Discriminator is recognized by o1
UP-element is recognized by the carboxy-terminal domain of RNAP alpha-subunit (not recognized by o^70 but the polymerase itself)
Prokaryotes: Transition to open complex 1 - structural changes
At this point, the sigma-factor and RNA polymerase are bound to the promoter. In the closed complex, o1.1 is located between the B and B’ clamps to prevent unwanted closing of RNAP, as well as collapse of RNAP. Hence, o1.1 functions as a gatekeeper between these two subunits of the polymerase.
During the transition to the open complex, 2 things happen:
- o1.1 is removed from the BB’ claw, leaving place for the DNA to enter into the polymerase
- BB’ claw clamps down and secured the DNA, allowing a transcription bubble to be formed
Prokaryotes: Transition to open complex 2 - transcription bubble, isomerization
o2 is responsible for the opening of the transcription bubble, and can do this in the absence of energy. The transcription bubble forms at the -10 element between -10 and +2 of the promoter.
o2 contains two pockets: one for adenine at -11 and one for thymine at -7 of the -10 element. These nitrogen bases are isomerized (flipped) into these pockets. As a result, there are less hydrogen bonds between the complementary bases at -10, which weakens the interaction between the two strands of DNA -> Melting of the dsDNA to ssDNA.
Characteristics of the isomerization process: Spontaneous and irreversible
Prokaryotes: Initially transcribing complex = Abortive transcription
Upon RNAP entering elongation, there is a production of several <10 nt-long abortive RNAs. Unknown reasons, unclear biological significance. To do so, the polymerase doesn’t start traveling along the template strand, but stays where it is. A process called scrunching involves bringing the DNA to itself, as the template is squeezed inside the active site. Afterwards, the abortive RNA is released. Scrunching happens several times, but once the polymerase finally manages to synthesise a longer RNA strand, the elongation process starts.
Prokaryotes: Promoter escape
First step of elongation - the RNA polymerase has to stop interacting with the transcription factors. Once the nascent RNA has reached 10 nt, the RNAP dissociates from the sigma-factor.
Prokaryotes: Elongation (incl. proofreading mechanisms)
Elongation includes the assembly line (polymerase channels, phosphodiester bond, polymerization reaction along the template strand). rNTPs and downstream DNA enter via dedicated channel. One rNTP is added at a time by a new phosphodiester bond, followed by translocation of RNAP to the next nucleotide. RNA and upstream DNA exit via dedicated channels.
There are two proofreading mechanimsms in bacteria for removing misincorporated nucleotides:
- Pyrophosphorolytic editing: Catalytic activity in reverse. When RNAP detects a ribonucleotide that shouldn’t be incorporated, it carries out the formation of the phosphodiesterbond in reverse to “erase its work”.
- Hydrolytic editing: RNAP backtracks or reverse translocates by cleaving the wrong nucleotide. Backtracks one nucleotide until it reaches the error, and cuts the phosphodiesterbond here.
Prokaryotes: Transcription-coupled DNA repair
In addition to misincorporated nucleotides, something else during elongation that may bother the RNAP is DNA damage in the shape of lesions. This makes the RNAP stop transcribing, and will also create a roadblock for other transcribing RNAPs (many polymerases transcribing the gene at the same time, traffic).
Some factors such as TRCF will remove the stalled RNAP in an ATP-dependent manner. It will also recruit nucleotide excision repair enzymes (NERs) that will remove the DNA lesion and allow transcription to continue normally.
Prokaryotes: Termination (two mechanisms, not in-depth)
Termination refers to the dissociation of RNA, DNA and RNAP. Occurs at the end of the gene at sequences called terminators, via two different mechanisms in bacteria:
- 50% Rho-dependent
- 50% Rho-independent
Prokaryotes: Rho-dependent termination
In bacteria, transcription and translation occur simultaneously - as soon as mRNA exits the polymerase, it is bound by ribosomes that will start translation (translation is co-transcriptional). Rho is a homohexamer with a O-ring shape that can bind to nascent RNA only in absence of ribosome at a Rho binding site called Rut (transcribed recently by the polymerase). When RNAP arrives at the end o the gene, there are no ribosomes there, and the RNA has been fully translated -> Rho can bind in an ATP-dependent mechanism, placing the RNA in the middle of the ring shape -> Rho travels along the RNA and pushes the RNAP away (through a dramatic change in conformation) -> The whole complex dissociates.
Prokarytes: Rho-independent termination
This mechanism is based on a different type of termination sequence - a specific terminator. The terminator contains an inverted repeat of ca. 20 nt (dyad symmetry, reverse of each other) followed by ca. 8 A:T basepairs (A:T rich region).
When transcribed, the inverted repeat basepairs to form a hairpin (because they are complementary to each other). Thought, like Rho, to trigger conformational changes in the polymerase which triggers dissociation from the DNA and RNA. Similarly to Rho-dependent mechanism.
The A:T rich region gets transcribed into a poly-U-tract -> When it is still annealed at the transcription bubble, it makes some A:U hydrogen bonds. These bonds are weaker than C:G or A:T, and consequently they will facilitate the release of polymerase from the transcription bubble (dissociation of RNA:DNA duplex).
What are some similarities and differences between transcription in bacteria and eukaryotes (from table)?
Similarities:
- RNAPs structure is similar (crab claw)
- RNA synthesis reaction (polymerization) is identical
Differences:
- RNAP number: 1 vs 3 (5 in plants)
- Initiation factor: Sigma-factor vs 6 general transcription factors (GTFs)
- gDNA template: Naked vs Chromatin (nucleosomes)
- Additional factors: Few vs Many
Eukaryotes: What is the name of the complex that recognizes the promoter so that initiation can occur?
PIC: Pre-initiation complex (of transcription)
Eukaryotes: What is the core promoter?
Core promoter consists of a minimal set of DNA sequences required for transcriptional initiation by RNAP II. Typically ca. 40-60 bp long, located upstream or downstream of TSS.
Eukaryotes: Characteristics of RNAP II core promoters
Different core promoter elements which are recognized by different transcription factors.
Most frequent core promoter elements/sequences:
- BRE: TFFIB recognition element - upstream of TSS / +1
- TATA box: TBP (TATA box protein) recognition element - upstream of TSS / +1
- Initiator (InR): TFIID recognition element - overlapping with the TSS / +1
- DPE, DCE, & MTE: TFIID recognition elements - downstream of TSS / +1
These elements are never found together at the promotors, only a subset. Most of the times you will find either InR + TATA box, or TATA box and DPE (two most common combination)
Eukaryotes: The 6 general transcription factors (GTFs) + name and functions
TFIIA: Stabilization of TBP at TATA box.
TFIIB: Stabilization of TBP at TATA box (grasping around TFIID) at both sides. Binds to BRE_u and BRE_d near the TATA box, detects the TSS, and ensures the direction of transcription.
TFIID: Large multi-subunit complex formed by TBP (TATA binding protein) and 13 TAFs (TBP-associated factors). TBP recognizes the TATA box of the core promoter -> Interacts with the minor groove -> Triggers strong bending of DNA at TATA box. The TAFs, especially TAF1 & TAF2, recognizes the initiator region. TAF6 and TAF9 will recognize DCE and DPE.
TFIIE: Promotes recruitment of TFIIH.
TFIIH: Extremely important multisubunit GTF, consisting of 10 subunits. Consists of 2 modules: 1) Core of TFIIG which contains XPD subunit (enzyme with translocase activity) and 2) CAK which contains a protein kinase called CDK7 (with kinase activity). The modules are linked by the XPD subunit (also has helicase activity, not important now). TFIIH is responsible for the formation of the open complex, as well as promoter escape.
TFIIF: Recruited together with RNAP II. Formation and stabilization of the closed complex.
Eukaryotes: Steps of PIC assembly at the core promoter
- TBP subunit of TFIID recognizes TATA box on the promoter -> Strong bending. TAFs of TFIID recognizes INr, DCE and DPE.
- Stabilization of TBP at TATA box by TFIIA and TFIIB. TFIIB is also in charge of detecting the TSS and ensuring correct direction of the transcription.
- Recruitment of TFIIF and RNAP II -> Formation and stabilization of the closed complex (the DNA is closed)
- Recruitment of TFIIE, which promotes recruitment of TFIIH. TFIIH has translocase and kinase activities, and is responsible for formation of the open complex, as well as promoter escape.
Eukaryotes: Transition from closed to open complex (by TFIIH) + abortive initiation
Achieved by the XPB translocase activity in TFIIH. While bacteria has a sigma-factor which can open up the DNA at -10 element in the absence of ATP, TFIIH translocase activity of XPB requires using the energy of ATP hydrolysis.
TFIIH is recruited and binds at the core, and the translocase activity pushes the DNA until it is opened -> Formation of transcription bubble, stabilized by other GTFs -> PIC ready for initiation.
Like in bacteria, initially transcribing complex involves abortive mRNA synthesis and scrunching in several rounds before elongation starts.
Eukaryotes: Promoter escape
Once the initially transcribing complex is formed, the polymerase must escape the promoter and start elongation by entering the gene body. TFIIH is also involved here
RNAP II has two large subunits: RPB1 and RPB2. The largest, RPB1, has a long C-terminal domain (CTD) which is extremely long and repetitive. CTD repeats: 52 (in human) or 26 (yeast) heptapeptides Y1S2P3T4S5P6S7. The more complex the organism, the more repeats there are.
Two of the amino acids among the CTP repeats, serine 2 (S2) and serine 5 (S5) are targeted by protein kinases (incl. CDK7 in TFIIH) and phosphorylated -> Become phospho-serine2 and phospho-serine5. The RNAP II is originally recruited to PIC unphosphorylated. CTD phosphorylation at serine 5 by CDK7 in TFIIH -> Signal for the RNAP to dissociate from the PIC / GTFs and escape the promoter. The phosphorylation is also involved in mRNA capping.
P-TEFb phosphorylates serine 2 (involved in elongation)
Eukaryotes: Elongation
The exact same process as in bacteria: Assembly line, rNTPs recruited to the RNAP, complementary base-pairing, phosphorylation etc.
There is a switch in partners from promoter escape and elongation. Before promoter escape, the RNAP was interacting with initiation factors -> TFIIH prevents this, and the polymerase leaves them behind. Now it interacts with elongation factors and RNA processing factors
RNAP II associates with:
- CTD phosphorylation at serine 2 by P-TEFb
- Elongation factors that stimulate elongation
- RNA processing factors
Eukaryotes: P-TEFb
One of the most important (positive) transcription elongation factors, together with FACT. Protein with kinase activity.
Functions:
1) Phosphorylates CTD serine 2 -> Promotes recruitment of splicing enzymes
2) Phosphorylates NELF and DSIF after mRNA capping -> Promotes RNAP II pause release. Right after promoter escape, the RNAP doesn’t immediately travel to the gene body until the TTS. Rather, stops 20-60 nt after the TSS because the mRNA strand needs to get capped. Pausing leaves time for the RNA capping machinery to perform its job. Pausing involves two proteins: NELS and DSIF, which are responsible for mRNA capping. When these are phosphorylated, the pause is released and the transcription can continue.
Eukaryotes: FACT (FAcilitates Chromatin Transcription), two-step mechanism
The other most important transcription elongation factors, together with P-TEFb.
Chromatin impedes transcription in eukaryotes -> When RNAP travels along the gene, it wncounters nucleosomes which the DNA is tightly wrapped around -> RNAP is prevented access to the sequence. RNAP must displace nucleosomes as it transcribes along the gene.
FACT helps RNAP II displacing nucleosomes (loosening up the chromatin ahead of the polymerase). FACT consists of 2 subunits, SPT16 and SSRP1, which binds to each of their histone dimer subunits. SPT16 binds to histone dimers H2AH2B, while SSRP1 binds to histone dimers H3H4.
The displacement of nucleosomes is a two-step mechanism:
1) FACT removes H2AH2B ahead of RNAP II -> RNAP II can progress (open transcription bubble)
2) FACT repositions H2AH2B after RNAP II has passed -> Maintenance of chromatin integrity.
Eukaryotes: Elongating RNAP II recruits RNA processing enzymes - capping, splicing and polyadelynation (overview)
RNAP is recruited to the promoter unphosphorylated. It is then phosphorylated at serine 5 by TFIIH, which triggers promoter escape and capping. Then it is phosphorylated at serine 2 by P-TEFb during elongation, which promotes slicing and polyadenylation.
Phosphorylated CTD serves as docking site for recruitment of RNA processing factors during elongation.
- Capping: Phospho-Serine 5 recruits capping enzymes -> Transferred to nascent RNA to create a methyl cap.
- Polyadenylation: Phospho-Serine 2 recruits splicing and polyadenylation enzyme -> Recruited to nascent RNA, and process it.
Eukaryotes: mRNA capping
The very first RNA processing mechanism occuring during transcription and elongation.
Capping enzymes are recruited by CTD phospho-serine 5. 60 nucleotides after the TSS, RNAP stops to allow this mechanism to happen = RNAP II pausing. Result: Addition of a G (guanine) methylated at position 7 (mg7) at the 5’ of nascent RNA, which comes out of the RNAP after it starts transcribing. It has 3 phosphates here, where there will be 3 main reactions
1) RNA triphosphatase removes the first phosphate group (gamma), left with two phosphates (alpha and beta)
2) Guanylyltransferase makes a bond between the two phosphates and the phosphate og guanine, forming a 5’ - 5’ triphosphate bond (very unusual).
3) Methyltransferase transfers a methyl group to the position 7 -> Formation of a m7 guanine cap.
The methyl cap now protects the nascent RNA from degradation by exonucleases (like a shield). It also regulates of nuclear export and promotes translation.
Eukaryotes: mRNA polyadenylation (unique to RNAP II)
CTD phospho-serine 2 recruits polyadenylation enzymes CstF and CPSF. At some point right before termination, the RNAP will encounter a specific sequence called the poly-A signal. Poly-A will be transcribed by RNAP and end up in the RNA, which triggers the transfer of polyadenylation enzymes from CTD to RNA where they bind to the poly-A signal. CstF and CPSF cleave the nascent RNA from RNAP II.
The problem is that the end is not protected against degradation (could be target of RNA degrading enzymes) -> Enzyme called poly-A-polymerase (PAP) and additional poly-A-binding factors create a tract of As (a poly-A-tail) which will be bound by poly-A-binding proteins. The poly-A-tail will protect from degradation by exonucleases at 3’ end, providing the mRNA strand with one shield at each end.
Eukaryotes: Termination - the torpedo model
Highly unclear process observed in mammals, widely accepted.
RNAP II does not stop transcribing after cleavage and polyadenylation of mRNA, but continues transcribing several Kb of DNA before termination. It has released the product, but continues working on the DNA to produce a second uncapped RNA (vulnerable to degradation). Enzyme called Rat1 (yeast) or XRN2 (humans) are recruited to degrade the RNA as it comes out of the polymerase as a torpedo -> Degradation is very fast and potent -> Collision between Rat1 and the polymerase (similarly to Rho in bacteria) which triggers conformational changes (pushing it forward) that dissociates the RNAP from the DNA and second RNA.
Eukaryotes: Difference between the 3 RNAPs
Similar in structure and share some subunits, but use different core promoters and work with different GTFs (but all use TBP)
Eukaryotes: Transcription by RNAP1
Transcribed ribosomal RNA (rRNA), which is the most abundant RNAs in the cell (ca. 80% of total RNA). rRNAs are encoded by one gene that exists in ca. 200 copies -> The RNAP I is working really hard for this production.
Initiation involves a core promoter and an upstream control element (UCE). UCE functions as binding site for protein called UBF, which recruits SL1 (consists of TBP and 3 TAFs) and RNAP I -> Transcription.
Eukaryotes: Transcription by RNAP III
Transcribed tRNA genes, 5S rRNA and a few other non-coding RNAs. Four types of core promoters -> Most of the tRNAs (between 100-300 genes) have the tRNA gene promoter.
This core promoter is formed by Box A and Box B downstream of TSS, meaning that the whole promoter is within the gene body. These two boxes are recognized by a general transcription factor called TFIIIC, which recruits TFIIIB that contains TBP. TFIIB then recruits RNAP III -> Transcription.
