Flashcards in Transcription Deck (81):
Information and the central dogma
• DNA as information storage
• mRNA as information carrier
– Produced through a process called transcription
– mRNA is a short-lived copy of the instructions carried in DNA (instances where mRNA lives longer in cell while they're bing translated
Messenger RNA - mRNA
Ribosomal RNA - rRNA
Transfer RNA - tRNA
MicroRNA-miRNA (gene expression)
Structure of RNA
Secondary structure can form hairpins
Secondary structures in mRNA can control how transcription can end or how translation can start
Transcription in Prokaryotes
• All RNA’s are made by transcription
• Template - DNA, DNA dependant RNAsynthesis
• Enzyme - RNA polymerase helps form RNA DNA hybrids
• Substrates - Ribonucleoside triphosphates that are added in
• Only one of the two strands are copied into RNA for a given gene
What is special about the first base added in on the hybrid?
RNA pol does not require hydroxyl group to add a base. As long as RNA pol is bound to DNA at a specific part of the enzyme, the complimentary RNA base will be incorporated in on the DNA template, and then every base from then on can use that 3' hydroxyl. First base is special, added based on complimentary base pairing
Additionally, keeps its three phosphates
Which DNA strand is copied?
Template strand is what RNA is made from. (Antisense). mRNA that is formed has same base or as sense strand except with Us instead of Ts
What do 5' and 3' refer to?
Carbon positions on the sugars of the ribonucleotides
Need to go this way because of the hydroxyl on the 3' carbon
What kinds of bonds are formed during synthesis of RNA from DNA template?
Phosphodiester bond formed from RNA polymerase after base pairing occurs. Nucleophilic attack on the 3' OH
Hydrogen bonds between complimentary base pairs
The importance of base pairs
Patterns of inverse base pairs are different in the major groove
RNA polymerase associated with a protein called sigma
Active Site - Mg 2+
It has channels for the DNA to go through and for new RNA bases to come in, and mRNA to trail out
What are the five subunits of the holoenzyme?
σ factor recognizes the promoter, binds first
α2β2 subunits-constitutes the core polymerase that catalyzes synthesis of RNA
Where is the promoter region in prokaryotes?
-35 and -10 nucleotides upstream from the start site
These consensus sequences are conserved
What does sigma do?
Recognizes promoter region (usually sigma 70)
How did researchers discover what sigma does?
• When researchers mixed RNA polymerase, sigma and DNA together in cell free cultures (lysed cells) they found that the holoenzyme would bind only to specific sites on the DNA
• Sigma is a regulatory protein
they didn't include sigma, they didn't get the same results. Sometimes enzyme would associate, but not as strong.
Untranscribed region, upstream of start point
Where the first RNA base is added
Specificity of sigma factors
Some sigma factors factors that are specific for certain genes, and those genes will only be transcribed when sigma factor is present.
Steps in prokaryotic initiation:
• Formation of the “closed complex”
• Unwinding of DNA to yield the “open complex”
• Synthesis of 5-10 phosphodiester bonds
• Release of sigma factor- once the RNA begins to form it “outcompetes” with sigma factor and it is displaced
Formation of "closed complex"
At first there is a loose association between DNA and core enzyme, it is positioned a site where first base will be added (but RNA chains that begin are not located at the proper sites)
Closed complex- when sigma and RNA polymerase enzyme bind to the promoter region.
Unwinding of DNA to yield the “opencomplex”
Sigma opens the DNA helix and transcription begins
Small open complex formed. Its reaction is exergonic.
Two hypotheses for initiation
One hypothesis is that sigma associates first and then scans for the promoter region.
The second hypothesis is that sigma binds to the promoter and then it brings in the polymerase and then the holoenzyme is formed on the DNA (accepted)
The start of RNA
RNA is initiated with the binding of two rNTPs (ribonucleotide triphosphates) and the formation of the first phosphodiester bond.
Synthesis of 5-10 phosphodiester bonds
5-10 bases added in, and the the DNA template's affinity for RNA polymerase goes up, and it is no longer associated with sigma as tightly and sigma will fall off.
After about 10 base pairs the holoenzyme will disassociate to a degree because the sigma factor will no longer be associated with the polymerase
Helps orient the two DNA strands back together
helps separate the DNA bases as the mRNA is being made, and stabilizes ssDNA
Rho (a protein) dependent or independent
The termination site is heavy in As, which forms Us on 3' end of mRNA
A stem loop (hairpin) is formed that is a palindromic sequence
Only 8 or 10 nucleotides in the RNA DNA hybrid, and Au base pairs (RNA DNA hybrid) weakly associated and the hair pin is stable
hair pin is in the exit channel in the polymerase which stalls the polymerase and causes it to disassociate.
What are the factors in disassociation at the t site in prokaryotes?
Stability of hairpin
Where it ends up in the polymerase (exit channel)
Weakness of AU bases
Rho dependent termination
Still form hairpin that disrupts polymerase that is stuck in exit channels that stalls polymerase.
During that, rho protein encircles mRNA and spins around it trying to catch up to the polymerase and when the polymerase stalls, rho can catch up. It has helicase activity and it helps to disassociate the DNA RNA hybrid, helps to pull off mRNA.
Rho and pol fall away
Similarities in prokaryotic and eukaryotic transcription
• Both processes go from 5’ to 3’
• Transcription initiates at a promoter
• Both utilize RNA polymerases
• Regulation of transcription initiation is the most common mechanism for control
• Both involve other transcription activators and repressor proteins that bind to specific DNA sequences and influence transcription rate
Differences between prokaryoticand eukaryotic transcription
• Eukaryotes have 3 different RNA polymerases
• RNA polymerases have more subunits in euk
• Prokaryotic promoters are recognized by a subunit of the polymerase. In eukaryotes, the core promoter often contains aTATA box at -30 that is recognized by TBP. TBP recruits the RNA polymerase
• For prokaryotes “promoter” refers specifically to the RNA polymerase (Holoenzyme) binding site. In eukaryotes, this refers to all of the protein recognition sites
• In eukaryotes there are many more transcription factors.
The initial RNA transcript (primary transcript, HnRNA (hetero nuclear RNA)) is clipped apart and spliced back together!
The mRNA can be as small as 10% of the size of the primary transcript, there are exonsand introns in eukaryotic genes
Lots of regulatory elements
• Pol I - transcribes ribosomal RNA
• Pol II - transcribes protein coding genes
• Pol III - transcribes tRNA and other small RNA’s
All of these contain two large subunits and 12-15 smaller subunits
How did researchers discoverthe different polymerases?
• Death cap mushroom and alpha amanitin
• Different concentrations had different effects.
• Low Pol II
• High Pol III
Low concentrations of the poison, pol II activity stops. mRNA can't be made. Cells can't make proteins, some of those proteins detoxify poisons. Also affects pol III, tRNAs.
Eukaryotic mRNAs are produced in distinct steps
• Synthesis of a primary transcript
• Modification of the 5’ and 3’ ends of the transcript
• Introns are spliced from the primary transcript
• Remaining coding sequence is comprised of exons
all of introns and some exons removed, gene can code for more than one protein
RNA pol II transcription unit
Unicellular: Upstream activating sequence
Multicellular: Long range regulatory elements(insulators and enhancers), can be 700-1000 bp away
They have to be in cis because enhancer elements are on the DNA. Has to be in line with that coding sequence.
Trans regulatory elements can be diffusable- move around in the nucleoplasm Ike RNA pol II.
Enhancers can be anywhere in the sequence
Euchromatin is transcriptionally active while heterochromatin is not (Some DNA is silenced because it remains tightly bound to the histones)
Insulators keep transcription machinery from being fooled if its surrounded by heterochromatin. There's no way for transcriptionally inactive DNA to inhibit transcription of eukaryotes
Some DNA is silenced because it remains tightly bound to the histones
Steps in Initiation for eukaryotes
• Binding of general transcription factors and RNA polymerase II to promoter
• Unwinding of DNA
• Phosphorylation of carboxyl terminus of RNA polymerase II (Post translational modification)
• Synthesis of first 5-10 phosphodiester bonds
• Release from promoter and most general transcription factors.
Assembly of Active Transcription Complex
– General Transcription Factors (TFIID, TFIIA, TFIIB) must bind before Pol II. (Accepted)
– Holoenzyme forms between Pol II and general transcription factors first and then locates promoter.
– located at about -30 relative to transcription initiation site
– Is bound specifically by TFIID
– Acts similar to a prokaryotic promoter to position RNA polymerase II for transcription initiation.
– located at +1
– bound by specific TAF (TBP associated factors)
– CpG promoters, ~20-50 CG repeats upstream in the promoterregion. Usually transcribed at a low rate. Cpg islands are GC rich. P is phosphate on sugar phosphate backbone. Sometimes island little upstream of the promoter
TFII (TBP and TAFs), and changes the shape of the DNA a little, signal is sent to other transcription factors
TFIIB (recruits RNAP II-TFIIF) and TFIIA (prevents inhibitor binding) bind to both DNA and TFIID TFIIA doesn't always bind
RNA Pol II- TFIIF binds (TFIIF is a bridge between proteins associated with recognizing the promoter region and RNA pol)
TFIIH binds (helicase activity)
TBP (TATA binding protein)
monomeric protein that is highly symmetrical and conserved between species (80% identical between yeast & humans in the conserved domain)
Has 8 beta sheets that line up and fit in the minor groove of DNA(recognizes TA or AT)
Sits like a saddle, and on coils like stirrups on either side of DNA. Causes sharp bend (almost 90 degrees) , change in shape of bps. At has added strain, easier to melt/break
C and N terminus are on the same side
How does TBP accomplish the bending?
• High levels of contact between DNA and TBP
• Positively charged lysine and arginine bind with the negative phosphates
• The kink in DNA is produced by phenylalanine residues projecting in between base pairs – further increases contact with DNA (think about TBPs shape
• Strain causes “melting” (what base pairs are at the binding site?)
RNA polymerase is modifiedduring transcription initiation
Doesn't get modified until it is associated with the DNA
• The carboxy terminal domain (CTD) contains 7 aa that arerepeated multiple times.
• Consensus sequence:
• Yeast: 26 repeats
• Humans: 52 repeats
• Ser and Tyr modified by phosphorylation
TFIIH and carboxy terminal domain kinase activity
Using ATP, phosphorylates carboxy terminus, change in conformation, energizes it and kicks off RNA pol II from the general transcription factors and helps it start elongation
Once RNA pol II is phosphorylated, it can unwind DNA, synthesize RNA, and proofread
Everything about phosphorylation
Carboxy terminal domain of II has heptad repeats (the amino acids that occur over and over) different positions on that repeat can be phosphorylated. Tf2h has kinase activity, phosphorylates AA 5 position. Then promoter escape- pol II kicked off, leaves promoter sequence. mRNA is made. Then 2 position is phosphorylated.
Phosphorylation helps initiate transcription and encourage other things that happen. Phosphorylated carboxy terminal domain supports capping enzyme. Supports splicing machinery
How does mRNA stay linear
RNA likes to form hairpins and loops but we need to splice out introns. Phosphate groups support protein machinery, allows mRNA not to assume loops and stay linear
Assembly of the Pre-Initiation Complex
1 Binding of TFIID (TBP plus TBP associated factors (TAF))
2 Binding of TFIIB (and sometimes TFIIA)
3 Binding of Pol II, TFIIF, TFIIE, TFIIH
4 Phosphorylation of CTD (carboxy terminal domain) ofPol II and unwinding of DNA
5 Formation of first phosphodiester bonds
What happens when RNA pol II leaves the promoter region?
Initial recognition proteins are left so new pol II can come. Increased amount of certain protein in cell results if many polymerases bind there
Eukaryotic Elongation factors
PTEFb- kinase, helps to phosphorylate c terminus
ELL and SII
Eukaryotic termination of transcription
Pol dissassociates from ell and sii, comes back to find another FIID bound to the DNA
FCP1- phosphatase, removes phosphates
Any pol 2 can bind to any promoter region that shows transcriptionally active
How do eukaryotic genes vary?
Size and complexity
Due to intron/exon ratio
Human factorVIII gene (blood clotting gene) 200,000 base pairs but barely any exons
Human beta globin gene- 2000 bp, barely any exons
RNA processing in eukaryotes
3' polyA tail
Eukaryotic mRNA’s have a 5’ cap formed by a 5’-5’ linkage of a 7-methyl GTP residue
Exonucleases in cell, eat from 3' to 5' but can't eat from 5' to 5', so cap resistant to exonucleases.
Also signal for transporting out of the nucleus
How can the 5' cap be used against your body?
One Family of viruses can take the cap and put in on the genome, but your own exonucleases can't attack cap
Why don't prokaryotes need the cap?
Transcription and translation occur in the same place in prokaryotes. Where in euk, mRNA has to be translated out of the nucleus, cap helps mRNA he carried out of the nucleus. Has to be put on in the nucleus because of the exonucleases in the nucleus
How is the cap made?
How was splicing discovered?
• Early experiments indicated that RNA was synthesized as a longer precursor.
• This larger class of RNAs was referred to as heteronuclear RNA or hnRNA (many different sizes)
• The mRNA found associated with polyribosomes was on average much smaller than the hnRNA.
• mRNA is derived from hnRNA
• mRNA’s are smaller in length than hnRNA’s, something must have happened
Other experiments showed:
The size difference between hnRNA and mRNA primarily due to removal of introns.
Length of mRNA
• Are mRNAs as long as the region of DNA encoding the gene?
• Use of DNA-RNA hybrids to see the splicing. RNA only base paired to the exons
Transcription and processing of the ovalbumin gene
Only 24% of the original transcript was left after splicing
Addition of poly A tail
• Specific signal sites dictate where cleavage proteins (endonucelase) and polyadenylation proteins (polyA polymerase) will attach. The upstream poly(A)signal is a specific sequence of nucleotides AAUAAA.
• The downstream poly(A) signal is a GU-rich region on the pre-mRNA.
• After transcription has been completed, the first two cleavage factors attach to the up and downstream poly(A) signal sites.
• PolyA polymerase associates with the cleavage proteins, cleaves the mRNA and then polyadenylation can occur
PolyA polymerase adds about 200 As to the end, protects 3' end of RNA from exonucleases. Needs to last long enough to go into cytoplasm and associate with ribosome
Does cleavage and polyadenylation always occur efficiently?
Needs two polyA signals
Upstream polyA signal: AAUAAA, can only mutate to AUUAAA, or polyadenylation is eliminated. Poly a tail wouldn't be added on. Polya polymerase recognizes this sequence
Function of 5’ cap and Poly-A
• Translation initiation
• Transport out of nucleus
– Prokaryotes mRNA’s degraded from 5’-3’ – In eukaryotes mRNA’s are degraded from 3’-5’
• Poly A tail can be used to purify mRNA
Wha sims the spliceosome made of?
SnRNPs (small nuclear ribonucleoprotein particles)- complex of RNA and proteins
Each snRNA is associated with 10 or more proteins
What are all the snRNPs called?
Rna components are Uridine rich RNAs, so they are Refferee to as U1, U2, U4, and U5 and U6 (U3 helps splice ribosomal RNA)
2 transesterification steps
• 2 Transesterification steps
– Branch-5’ splice site (formation of lariat)
– 5’ splice site-3’splice site
– Release of intron as lariat
How does spliceosome know where to go?
• First step is the recognition of the junction between the exons and introns.
• Recognition involves RNA-RNA base pairing assisted by proteins
What four conserved sequences are necessary?
– 5’ splice site, 9 bases
– branch point, UAAC
– pyrimidine track
– 3’ splice site, 5 bases
Formation of the spliceosome
RNA in these proteins recognize the splice site
1- Binding of U1 snRNP to 5’ splice site
2-Binding of U2 to branch point
3- U4/6 binds to U2, and U5 binds closer to 3' end (5' end of exon)
Procedure of splicing
Spliceosome cleaves the phosphodiester bond at the 5' end of intron
U1 is released
U4 is released, lariat is formed, and formation of phosphodiester bond between 5' G of intron and conserved A near 3 end of intron
3' site is cleaved and two exons are joined by phosphodiester linkage
Transcription and RNA processing are coupled
Phosphorylated CTD interacts with processing enzymes
Change in the function of CTD - think about the length of some mRNA transcripts
Stabilized by carboxy terminus domain, can be multiple spliceosomes at the same time
Group one introns
Require G factor
Group two introns
Similar to spliceosomal, but no spliceosomes. Self splice, requires bulging A, catalyze own transesterification reaction
another way that alternative mRNAs and proteins are made
Change in base can control what's left in and what's taken out
Differences between closely related cells and their Apolipoprotein DNA sequences due to editing
How does alternative splicing affect proteins?
Proteins from genes experiencing alternative splicing are very similar except in key regions that might affect ligand binding, location and activity.
Can do different things in different cells, closely related though
What is the advantage of having introns that need to be eliminated before the mRNA can be translated?
• Main rationale used to explain the existence of introns is that it has allowed evolution to proceed at an increased pace.
– Exons frequently encode different domains of a protein which can be combined via DNA rearrangements to generate new proteins relatively quickly (exon shuffling)
• Alternative splicing allows a variety of related proteins to be synthesized from a single gene
• Introns are thought to be a “safe place” for DNA breaks
NHEJ wouldn't be so bad if intron bases were lost, non coding
Mutations can occur where the introns become exonized and code for the protein making it have higher fitness