|Lecture Notes: 1 April
© R. Paselk 2006
Recall the RNA is:
- ribose based,
- requires A, C, G, and U (instead of T) as bases,
- is generally single stranded, though RNA molecules may have hair-pins and double-stranded regions (even so often have imperfect base-pairing).
Three types of RNA:
- Ribosomal (rRNA), which serves as a major component of the machinery of protein biosynthesis.
- Transfer (tRNA), which serves as a "mechanical" translation device to convert the four letter nucleic acid code to the 20 letter amino acid code.
- Messenger (mRNA), which contains the primary sequence information for the protein - acts as an information transfer tape.
All of the RNAs are complementary to a DNA strand, so the active DNA (the strand which is "read") doesn't code, is the non-sense strand. The sense strand of DNA, the strand which may be directly out as a peptide sequence, is thus present only to maintain the information integrity.
There are significant differences between the RNA polymerase systems of prokaryotes and eukaryotes. In particular the eukaryotic systems studied have different polymerases for the different RNA types, in addition to polymerases in mitochondria and chloroplasts, while a single polymerase serves for E. coli. We will look at the prokaryotic system first as the classic model, then come back to the eukaryotic system.
E. coli RNA Polymerase
RNA polymerase requirements:
- Template - prefers double stranded DNA, but can use single stranded.
- All four NTP's (as opposed to deoxyNTP's for DNA pol) - ATP, CTP, GTP and UTP (note that U substitutes for T).
Note that there is no primer requirement. (Fig 26-1a,b,c; p 1023)
The reaction proceeds 5' 3', with an attack of the 3'OH of the growing chain on the 5'--P of a free trinucleotide:
Note that this reaction will leave a triphosphate on the 5'-end of a new RNA molecule.
RNA polymerase structure
The functional enzyme exists in two forms:
- The Holoenzyme consisting of an 2' pentimer of 480 kD molecular weight, making it one of the largest known soluble enzymes, with a diameter of about 100 Å diameter. The holoenzyme is used to initiate RNA biosynthesis, after which the sigma subunit dissociates to give the core enzyme.
- The Core enzyme consisting of an 2' tetramer. The core enzyme synthesizes RNA. (Fig 26-6b, p 1028)
Three stages in RNA polymerization:
RNA polymerase binds to specific promoter base sequences of about 40 nucleotides on the 5' side of the start site, n = +1. (Note that sequences are labeled as -n...-1, +1...+n where the (-) indicates the 5' side and (+) indicates the 3' side, and there is no 0.)
- The core enzyme binds tightly, but non-specifically, to duplex DNA. Addition of the sigma subunit gives the holoenzyme (Fig 26-7, p 1028), which binds only loosely to duplex DNA, allowing the the enzyme to diffuse along the DNA strand until it reaches a promotor region. The holoenzyme then binds tightly a region from about -20 to +20. Within this region is a consensus sequence around -10, referred to as a Pribnow box (= TATAAT, where TA ...T is highly conserved). Additional less conserved sequences occur at about - 35 and + 5-8. These vary the efficiency of transcription (dependent on the formation of an efficient complex).
- The holoenzyme binding to the promotor region results in a closed complex. This complex isomerizes, causing promotor to "melt", forming a "bubble", from -9 to +2 (the AT-rich sequence which has a lower binding energy, with its fewer H-bonds, than a mixed or GC rich sequence, making this opening easier).
(text Figure 26-6a-1, p 1000)
- The new RNA sequence generally starts with a purine, usually A, giving a 5' triphosphate: pppA + pppN pppApN + PPi. The polymerase synthesizes a "primer" of about 10 nucleotides which forms one turn of double helix with the DNA template, and the sigma unit is released. At this point the enzyme changes from initiation to elongation mode.
The initiated RNA strand now grows 5' 3'.
- During elongation the RNA polymerase core enzyme contacts about 35 nucleotides of DNA , of which perhaps 17 DNA base-pairs are unwound (opened), and about 11 are base-paired to the new RNA strand (approximately one turn of the helix).
- As synthesis proceeds we immediately run into a problem. The DNA is a coil, so either the polymerase must run around the coil, or the DNA must spin if it is left intact. Neither is likely since both molecules are massive and embedded in a high viscosity medium. To overcome this situation, topoisomerases are needed, one before the transcription bubble to relax the supercoiling as the bubble forms, and one behind to relax the supercoilling the other direction as the helix reforms. The two nicks will also allow the DNA bubble region to "spin" and the RNA will thus not have to wrap around the DNA helix.
Again a complex and multi-step process. RNA polymerase is a key player in termination, where the subunits can both increase and decrease the efficiency of termination. There are two types of termination, one dependent on another protein , the rho (rho symbol) factor, and the other on specific termination sites in the DNA of E. coli.
In the case of Termination sites there is not a unique base as a stop point. Rather some common structural features occur which result in termination:
- A series of 4 - 10 consecutive A-Ts with A's on the template strand - RNA termination occurs in or just past this sequence.
- A G+C rich region with a palindromic sequence immediately proceeds the A-T sequence. The RNA transcribed from the G-C rich sequence will be able to form a hair-pin structure (due to the self-complementarity of the palindromic sequence).
(text Figure 26-8, p 1029)
- A series of 6-8 A's on the DNA template strand, coding for U's in the RNA, which will bind to the DNA template only weakly.
The result of this termination site is that the RNA transcript will form a hair-pin which in turn slows the RNA polymerase. Since the last RNA synthesized, and involved in the RNA-DNA double-strand, will be the poly-U stretch, the polymerase and RNA will tend to peal off, as the somewhat more stable A-T bonds displace the A-U bonds, halting synthesis.
Rho factor () enables non-spontaneously terminating sites to terminate and increases the efficiency of the spontaneous termination sites discussed above. The rho factor is an ATP dependent helicase, which can unwrap the DNA-RNA hybrid helix. This ring-shaped hexameric protein (six 50 kD subunits) binds to the nascent RNA strand at a C-rich recognition site, then migrates 5' 3' towards the polymerase. When the polymerases 'pauses' at a G-C rich termination region, catches up, unwinding the RNA-DNA double helix and releasing the RNA polymerase resulting in termination.
Last modified 9 April 2009