Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 1 April

© R. Paselk 2006
 
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Recall the RNA is:

Three types of RNA:

  1. Ribosomal (rRNA), which serves as a major component of the machinery of protein biosynthesis.
  2. Transfer (tRNA), which serves as a "mechanical" translation device to convert the four letter nucleic acid code to the 20 letter amino acid code.
  3. 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:

Note that there is no primer requirement. (Fig 26-1a,b,c; p 1023)

The reaction proceeds 5'right arrow 3', with an attack of the 3'OH of the growing chain on the 5'-alpha-P of a free trinucleotide:

structural daigram of RNA polymerase reaction

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:

Three stages in RNA polymerization:

  1. Initiation,
  2. Elongation,
  3. Termination.

Initiation

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.)

Elongation

The initiated RNA strand now grows 5'right arrow 3'.

Termination

Again a complex and multi-step process. RNA polymerase is a key player in termination, where the beta 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:

  1. A series of 4 - 10 consecutive A-Ts with A's on the template strand - RNA termination occurs in or just past this sequence.
  2. 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)
  3. 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 (rho) 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'right arrow 3' towards the polymerase. When the polymerases 'pauses' at a G-C rich termination region, rho catches up, unwinding the RNA-DNA double helix and releasing the RNA polymerase resulting in termination.


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Last modified 9 April 2009