1. Transcription and message processing
Chemistry 256

2. Ribonucleic acids are an intermediary between DNA and proteins
Three principal types of RNA present in cells:
Ribosomal RNA (rRNA)
Messenger RNA (mRNA)
Transfer RNA (tRNA)
Within each of these types, though, the RNA molecule is processed (typically, edited and shortened).

3. Prokaryotic transcription
Samuel Weiss (Univ. of Chicago), “A Mammalian System for the Incorporation of Cytidine Triphosphate into Ribonucleic Acid”. J. Am. Chem. Soc. (1960) and Jerard Hurwitz (New York Univ.), “The enzymatic incorporation of ribonucleotides into polyribonucleotides and the effect of DNA”, Biochem and Biophys Res Commun (1960).
Hydrolysis of pyrophosphate drives the synthesis.

4. RNA polymerase has a “crab claw” shape
Isolated from Thermus aquaticus (Taq), the active site is between the red and blue “pincers”.
Structure elucidated by Seth Darst’s lab at Rockefeller University (Murakami et al. (2002), “Structural basis of transcription initiation: An RNA polymerase holoenzyme-DNA complex”, Science 296)
The “pincers” space is 27 Å when “open”; upon binding to DNA uses another subunit to narrow by 10 Å.
Uses Mg2+ in active site.

5. Formation of the RNAP holoenzyme
The core RNA polymerase (the Taq fragment) is a protein complex consisting of 5 subunits (two alpha, beta, beta', and omega). A sixth, modular subunit known as the sigma factor is required to recognize the promoter, melt the dsDNA, and provide a single stranded DNA template for transcription. When the sigma subunit attaches, the complex becomes the RNAP holoenzyme.

6. The transcription initiation process
The “pincers closed” complex has UP (promoter) element contacts, and thus is important in initiation as the DNA enters the channel. In the “pincers open” complex, the template strand of transcription bubble is in a tunnel formed by the ’subunits lined with basic amino acids, and gives rise to RNAP’s processivity.
The  subunit dissociates upon the elongation phase of transcription, even though the dissociation constant is quite small. There are several interactions between  and the DNA strand, none of which is particularly strong; as elongation occurs, some of these interactions are suppressed which allows  to fall off.

7. Not both of the DNA strands are “read” by RNAP
The 5’  3’ strand is called the “sense” strand; the 3’  5’ strand is called the “antisense” strand – but how can you tell which is which? Somewhat confusingly, the template for RNA synthesis is the antisense strand, because the actual coding bases for protein synthesis will be complementary to the noncoding DNA strand.

8. Gene organization
Genes that code for proteins are structural genes; in eukaryotes, they are transcribed individually. In prokaryotes, structural genes are organized into operons, and transcribed at the same time.
Lac operon contains genes that allow digestion of lactose.

9. Prokaroyte genes are preceded by a promoter sequence
The more closely the promoter resembles the consensus sequence, the stronger the promoter
David Pribnow (Harvard Univ.), “Nucleotide sequence of an RNA Polymerase Binding Site at an Early T7 Promoter.” Proc. Natl. Acad. Sci. USA (1975)

10. RNA strand grows by nucleophilic attack
The 3’-OH on the RNA strand attacks the α phosphate on the NTP.
Powered by the hydrolysis of the released pyrophosphate

11. DNA is unwound during transcription
One turn is removed to permit the binding of RNAP.
As RNAP advances, DNA is overwound in front (3’ end) and underwound behind.
Though the RNA-DNA hybrid forms a double helix, the RNA strand is stripped off as the two DNA strands reanneal (cf. DNA replication where the copies strands stay annealed to their respective templates).

12. Abortive initiation
If the transcription “bubble” ever gets too large, the conformational strain will “pop off” the RNAP (RNAP will not stay bound to the promoter) and end transcription after only 9 to 11 nt have been transcribed.

13. DNA polymerase – RNA polymerase collisions
DNA polymerase is faster than RNA polymerase, so two scenarios:
DNA P overtakes RNA P – like an express overtaking the local, RNA P continues to process a DNA strand even as it is being replicated (no displacement!).
DNA P head-on into RNA P – RNA P switches to a siding (daughter template strand).

14. RNAP is processive and quick
RNAP acts as its own sliding clamp and transcribes about 2000 bases before slipping.
In E. coli, the rate of transcription is about 20 – 50 nucleotides/second (slower than replication)
Error rate is one in every nt transcribed – no proofreading.
This rate is tolerated because most genes are repeatedly transcribed.

15. Transcription ends at termination sequences
Many E. coli genes share two features:
4 to 10 consecutive A-T base pairs with the As on the template strand.
A G+C rich region with a palindromic sequence that precedes the A-Ts.

16. Termination of transcription occurs due to stem loop formation
Knocks the RNAP “off track”.
In addition the poly-U tail downstream is displaced from the template strand by the non-template strand.

17. Other termination sites seem to require the “Rho” factor
Termination sites without the hairpin loop or poly-U tail require this hexamer to unwind the RNA from the DNA and thus free the RNA.
The binding sites for the RNA strand are shown in green and red below; rho requires a specific recognition sequence ( nt that lack a stable secondary structure and have multiple C rich regions, G poor regions) on the newly transcribed RNA upstream of the termination site.
Rho releases the mRNA transcript.

18. Some antibiotics inhibit prokaryote transcription
Actinomycin D binds to duplex DNA and inhibits replication and transcription; the phenoxazone rings are planar and intercalate with the DNA’s bases.
Two related antibiotics: rifamycin B and rifampicin
2 X 10-8 M rifampicin inhibits 50% RNAP
Binds to the  subunit and prevents chain elongation.
Box 26-2 figure 2

19. Eukaryotic transcription
Differs from prokaryotic transcription in having multiple RNAPs and more complex control sequences.
RNAP I (nucleoli) synthesizes precursors to rRNA.
RNAP II (nucleoplasm) synthesizes mRNA (shown below).
RNAP III (nucleoplasm) synthesizes tRNA, some rRNA and other short RNAs.

20. The mechanism of eukaryotic RNAP
Similar to prokaryotic RNAP, the key is the unwinding of the duplex DNA.
In the diagram, the duplex DNA enters from the right and is held in place by the “clamp”.
The duplex unwinds 3 nt before the template strand meets the RNA.
Much of this worked out in the lab of Roger Kornberg (Stanford), 1987 – present.
Box 26-2 figure 3

21. RNAP II is structured to separate duplex strands
The template strand meets the “wall” which causes a sharp turn in the direction of the strand movement.
At that point, the “funnel” directs NTPs toward the template strand (further aided by a well-positioned Mg2+ ion.
The growing RNA-DNA duplex forms a hybrid A-DNA/B-DNA structure, but the “rudder” separates the duplex.

22. Nucleotides are selected via two sites on RNAP
The E or entry site, which exhibits no base selectivity (looks for ribose, not deoxyribose).
The A or addition site, which checks the hydrogen bonding of the bases.

23. Accessory proteins help RNAP find promoter (initiation) sites
RNAP II (shown below) has multiple promoter sequences, each recognized by a different protein.
Note there are both downstream and upstream promoter elements; some are located at – 187 to –107.

24. Transcription factors (TF) are needed to start transcription
Highly-conserved proteins are necessary for allowing RNAP to start at the right position.
Along with RNAP and the DNA, these proteins form the preinitiation complex (PIC).

25. Transcription factors (TF) add in a very specific way
TF II D recognizes the TATA sequence and is part of the TATA-binding protein complex.
TF II A and TF II B recognize TF II D and orient RNAP properly.
TF II F brings in RNAP, then TF II E and TF II H act as energy sources and helicases.

26. There are a lot of transcription factors needed
Tupler et al. (2001), “Expressing the human genome”, Nature 409.
The listed domains and other entries are various transcription factors or regulatory portions of a genome.

27. TF II proteins play a major role in determining transcription direction
Since TBP can fit either direction on DNA, TF IIB orients it.

28. TF II also interacts closely with RNAP’s active site
Zinc finger (protein wrapped around a Zn2+ ion) of TF II enters active site, influences the nucleotides that are added onto the growing mRNA.

29. Eukaryotic mRNAs have a 5’ cap and a poly(A) tail
A 7-methylguanosine is added to the transcript’s initial (5’) nucleotide.
Compared to prokaryotes, eukaryotic termination sequences are imprecise, perhaps due to a transcript AAUAAA cleavage site.

30. mRNA synthesis removes many introns
Pre-mRNAs are processed by the excision of nonexpressed intervening sequences (introns), and the flanking expressed sequences (exons) are spliced together.
The introns are, on average, are four to ten times as large as the exons.
First intron was discovered in 1977 in adenoviruses; Richard Roberts (Cold Spring Harbor) and Phil Sharp (MIT) share Nobel in 1993.
Walter Gilbert coins the term “intron” in 1978.

31. Production of mRNA Primary transcript is capped and polyadenylated.
Excision of introns is performed.

32. Consensus sequence governs splicing of mRNAs
The pre-mRNA introns must contain “bounding” Gs. Note also the invariant U and A.

33. Transesterification reactions result in the splicing of exons in mRNA
Splicing begins with the phosphate at the 5’end of the intron reacting with an intron adenosine, and forming a lariat structure.
The left exon’s free 3’-OH reacts with the 5’ terminus of the right exon and shortens mRNA.

34. snRNP = small nuclear ribonucleoprotein
Highly conserved, nt RNA (left) forms a complex with Sm proteins (right).
A spliceosome (60S particle = RNA + protein) brings together pre-mRNA, snRNPs and other binding proteins (Steitz, 1980).
Structure indicates that the RNA moiety catalyzes splicing.

35. Alternative splicing can lead to protein diversity
Alternative splice sites can yield multiple proteins, as shown with α-tropomyosin below. Explains 100,000 genes hypothesized for human genome with 23,000 actually found.
SR proteins help direct which splice sites are active.

36. Different size rRNAs are made by shortening the initial rRNA transcript
Primary transcript is over 5500 nt long; longest rRNA is 2900 nt.
Enzymes RNase III, RNase P and others recognize secondary structures of RNA.

37. Methylation of 45S RNA allows creation of rRNAs
The exons (marked below) have numerous bases that are methylated.
These areas are then cleaved and cleaned up by Rnase III and Rnase P-like enzymes.

38. Small nucleolar RNAs (snoRNA) complex with proteins to modify rRNAs
snoRNAs are made from “discarded” introns complementary to the site where covalent modification of rRNAs take place.
snoRNA-protein complexes allow methyltransferase to methylate O2’ on rRNAs.
Some snoRNA-protein complexes modify uridine into pseudouridine.

39. Post-transcriptional processing of rRNA
Process to the right illustrates what happens in the protozoan Tetrahymena – first example of ribozyme.
Intron is excised by a self-splicing process that starts with a guanosine nucleotide, and ends with a cyclized intron.
Thomas Cech and others (Univ. of Colorado, Boulder), “Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena”, Cell (1982)

40. tRNAs are processed to yield a particular shape
In bacteria, tRNAs are self-splicing.
In eukaryotes and archaea, the same types of enzymes (endonucleases, ligases and phosphotransferases) that process rRNA work on tRNA to shorten it.

41. Post-transcriptional processing of tRNAs
tRNA must have two introns removed, the CCA “tail” added and various nucleotides covalently modified.
The ribozyme Rnase P performs this in both eukaryotes and prokaryotes – its RNA moiety contains the active site – true catalytic ability.