Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432

Biochemistry

Spring 2009

Lecture Notes: 6 April

© R. Paselk 2006
 
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Transcription, cont.

Eukaryotic RNA Polymerases

[See: Maniatis, Tom and Robin Reed (4 April 2002) An extensive network of coupling among gene expression machines. Nature 416 pp499-506 for new picture]

Unlike prokaryotes, eukaryotes have a variety of RNA polymerases: a mitochondrial polymerase (and a chloroplast polymerase in plants), and three nuclear polymerases. We will focus on the three nuclear RNA polymerases:

  1. RNA Polymerase I: This enzyme is localized in the nucleolus and is responsible for synthesizing the rRNA precursor.
  2. RNA Polymerase II: This enzyme is in the nucleoplasm, synthesizing the mRNA precursors.
  3. RNA Polymerase III: This enzyme is also in the nucleoplasm, but specializes in synthesizing tRNA, the 5s rRNA and other small RNA precursors.

There is much variety and complexity in the make-up of the three polymerases. All are large enzymes with up to 14 different subunits. Polymerase II, which is also know as RNA Polymerase B, has gathered the greatest attention as one would expect. A comparison of these enzymes based on Polymerase II from yeast follows. The subunits of polymerase II are named RPB1-10 (for RNA Polymerase B 1-10).

  1. RNB1 (220 kD): This largest subunit has homologous subunits with similar sequences in polymerases I & III as well as the E coli subunit beta'. It has an unusual structural feature not found in prokaryotes, a long C-terminal 'tail' (the CTD = C Terminal Domain) with 27 repeats of the sequence PTSPSYS (pro-thr-ser-pro-ser-tyr-ser). Note that this sequence is quite hydrophilic, and has many potential sites for phosphorylation (5/7 have -OH groups).
  2. RNB2 (150 kD): The next largest subunit again has homologous subunits with similar sequences in polymerases I & III and this time E coli subunit beta. As in the case of E coli this subunit binds a NTP. Both RNB1 and RNB2 participate in the catalytic site of the polymerase
  3. RNB3 (45 kD): The next largest subunit is homologous with the E coli subunit alpha. Two copies are present in the polymerase and are necessary for core assembly, as is the case in the bacteria. It is unique to Polymerase II in eukaryotes.
  4. RNB4 (32 kD): This subunit is the last to have a bacterial homolog, in this case sharing significant sequence similarity with the factor of E coli and thus thought to be involved with promoter recognition. It readily dissociates from the polymerase. Like RNB3, it is unique to Polymerase II in eukaryotes.
  5. RNB5 (27 kD), RNB6 (23 kD), RNB8 (14 kD), & RNB10 (10 kD) are all shared by the three eukaryotic polymerases.
  6. RNB7 (17 kD) Is unique to Polymerase II, and readily dissociates.
  7. RNB9 (13 kD).

Promoters and Enhancers

Eukaryotic polymerases differ in the strategies of promotion.

RNA Polymerase I

There is only one type of rRNA gene in a given species of eukaryote, though there may be hundreds or even thousands of copies of that gene. As a result there is only one promoter in each species for polymerase I, though the promoters are quite species specific.

The rRNA promoter for yeast has a sequence from -31 to +6 (core promoter element) with an additional upstream elements at - 187 and -107. A short sequence is probably required for polymerase binding with the rest required for transcription factors (Nested control regions).

The product of RNA polymerase I is a 7500 bp transcript (approx. 45s) which has, in order ( 5'right arrow 3') the 18s, 5.8s, and 28s rRNAs separated by spacers.

RNA pol II promoters are more diverse, as would be expected given the vast number of genes it transcribes.

RNA Polymerase II also has enhancers - sequences of variable portions and orientation relative to sequences - must be associated with promoters to function.

RNA Polymerase III

Promoters can be totally within transcribed sequences.

Translation

The Genetic Code

Major considerations in understanding the coding required to translate the four base nucleic acid alphabet to the 20 amino acid alphabet include:

In fact the code has proven to be a non-overlapping, non-punctuated, triplet code in which gene sequences are co-linear with peptide sequences, and where 5' right arrow 3' corresponds to NH2 right arrow COO-.

The code was originally elucidated in cell-free systems containing the complete protein synthetic system except for a messenger RNA (ribosomes, GTP, amino acyl tRNAs etc.). If polyU is then introduced to the system, a poly-phe is produced, so one codon for phe = UUU, similarly each of the other three polyNA's can be used. Then can do alternate (e.g. UCUCUCUCUCUC) two different amino acids will be coded etc. Finally, were able to synthesize and work with triplets to get the entire code.

The Code

The "Standard" genetic code is given in Table 27-7, p 1069 of your text. This is the code used by all known organisms, the only exceptions being some deviations in the mitochondrial tRNAs, and, it is now known, in the ciliated protozoa.

tRNA

tRNA functions as an adaptor to correlate the four base nucleic acid language to the 20 amino acid language. One end of the folded molecule binds to the three-base codon on the messenger RNA while the other end is bound to an amino acid residue. We discussed its structure earlier.

Aminoacyl tRNA Synthetases

Amino acid residues are covalently linked to tRNA in an "activated" form in a two reaction process:

  1. Activation of the amino acid residue:

    structural diagram of the activation of an amino acid by Aminoacyl tRNA Synthetases
  2. Formation of the aminoacyl-tRNA:

    structural diagram of the transfer of an activated amino acid to tRNA by Aminoacyl tRNA Synthetases

Note that the first reaction should have a free energy of about zero since we are breaking and forming acid anhydride bonds, and thus the reaction is driven by the subsequent hydrolysis of the pyrophosphate.

The second reaction is then driven to completion because the "activated" amino acid acid anhydride bond is broken and replaced with the relatively low energy ester bond.

Remarkably aminoacyl tRNA synthetases do not appear to be closely related to one-another (they have different sequences, and different folds!) - apparently they are so ancient they started independently. They exhibit a variety of quaternary structural patterns alpha, alpha2, alpha4, and alpha2beta2, with between 334 right arrow 1000 amino acid residues. As another indicator of the great age of these proteins, the aminoacyl tRNA synthetases for the same amino acids are similar in evolutionarily diverse organisms, but the aminoacyl tRNA synthetases for different amino acids in the same organisms are generally dissimilar.

In the case of tyrosyl-tRNA synthetase the catalysis appears to operate strictly via transition state and proximity/orientation catalysis - there is no classical chemical catalysis (acid/base, covalent, etc.) apparent.

Most aminoacyl tRNA synthetase-tRNA contact sites are on the inner face of the 'L', but otherwise show no regularity. Some seem to recognize only the acceptor region, others the anticodon, etc. (see figures 27-22 in your text).

Finally, aminoacyl tRNA synthetases exhibit remarkable specificity by the use of editing in addition to substrate binding. For example, for isoleucyl tRNA synthetase:

Wobble and Code Degeneracy

Even though there are isoaccepting tRNAs (different tRNAs specific for the same amino acid), it turns out that many tRNAs bind to a number of different codons specifying the same amino acid!

This observation is explained by the "wobble hypothesis" of Francis Crick. According to this model,

The various pairing possibilities allowed by wobble are shown in the table below (Table 27-4 in text):

Wobble Pairings

(third anticodon/codon positions)

5'-anticodon base 3'-codon base
C G
A U
U A or G
G U or C
I U, C, or A

The wobble hypothesis requires at least 31 tRNAs to translate all 61 coding triplets plus one for special initiation tRNA. Most cells have >32. All isoaccepting tRNAs in a cell have the same aminonoacyl tRNA synthetase.

Note that the most frequently used codons (those specifying the most frequently used amino acids) are complementary to the most abundant tRNA species.

Notes:


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Last modified 9 April 2009