Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 8 April	© R. Paselk 2002
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Transcription IV

Exons and Introns

As noted last time, the mRNA transcript, hnRNA is first processed in the nucleus by capping the 5' end and cleaving and adding a poly(A) tail to the 3' end. The is followed by additional processing in which the introns are removed before the final mRNA, capped and with a poly(A) tail is transported to the cytosol for translation.

Note that for most eukaryotic genes the majority of the transcribed RNA is never translated. Rather about 80% on average is excised as introns and degraded.

The process whereby the introns are excised leaving the exons strung together is referred to as gene splicing. Gene splicing is very precise (a single base error would result in an unreadable transcript) and assembles the exons in sequence.

There seems to be a very high degree of sequence homolgy at exon-intron junctions, which is necessary and sufficient for proper excision. In most eukaryotes these sequences include:

• There is an invariant GU at the intron 5' boundary.
• There is an invariant AG at the intron 3' boundary.

The actual excision of the intron occurs in two reactions:

1. A 2'-3' phosphodiester bond is formed by the attack of the 2'-OH on the ribose of a specific A and the 5' terminal phosphate, which releases the 5' end of the exon. As a result the intron gains a lariat structure on the 5' end. In vertebrates the A is within a highly conserved sequence, CURAY, located 20-50 residues upstream of the 3' end of the intron.
2. The resultant free 3'-OH group of the upstream exon now attacks the the 5' phosphate of the downstream exon, forming a new phophodiester bond and releasing the intron, and creating the spliced product.

The intron is released as a lariat structure, which is rapidly degraded.

Splicing is mediated by small nuclear RNA containing proteins (snRNPs or "snurps"). The small nuclear RNAs (60-300 bases) in these snRNPs are highly conserved. A number of snRNPs have known functions:

• U1-snRNPs RNA's 5' end is complementary to to a consensus sequence in the mRNA'a splice junctions.
• U2-snRNP recognizes the the intron lariat branch point.
• U5-snRNP recognizes the 3' splice junction.

The splicing itself takes place in the spiceosome particle. This large particle (50-60 s, about the size of the large ribosomal particle of E. coli = approx 1.6 megadaltons) includes a pre-mRNA, the snRNPs above and the U4-U6-snRNP (held together by base-pairing)and a variety of pre-mRNA binding proteins.

In addition to the modifications already noted, hnRNA also gets methylated to the extent of about 0.1 % of the A residues, of which many are retained in the final mRNA.

rRNA Processing

As we noted some time ago, rRNA in both E. coli and eukaryotes is coded in a large piece of RNA which must be leaved to release the large and small ribosomal RNAs and the 5s RNA of the ribosomes. Though, similar, the two systems differ in significant ways.

E.coli. There are seven polycistronic operons containing nearly identical rRNA genes and up to four tRNA genes each. The operon transcripts are cleaved by endonucleases.

• The rRNA primary transcript is first cleaved as it is transcribed by a series of specific endonucleases:
  • RNase III action gives the pre-16s and pre-23s rRNAs.
  • RNaseP, RNaseE and RNaseF action gives the pre-5s rRNA and some tRNAs.
• The 5' and 3' ends of the pre-rRNAs are then trimmed after they associate with ribosomal proteins to give the final rRNAs:
  • RNaseM16 trims16s rRNA
  • RNaseM23 trims 23s rRNA
  • RNaseM5 trims 5s rRNA

Eukaryotes generally have hundreds of tandemly repeated copies of the rRNA genes for the 5.8s, 18s and 28s RNAs.

• The transcript is arranged (5'-3') in the order: 18s, 5.8s, 23s with intervening spacers to give a 45s RNA of about 7500 nucleotides.
• The RNA processing is at least in part done via self-splicing (only a few have introns):
  1. The 3' OH of guanine forms a phophodiester bond with the intron's 5' end.
  2. The 3' OH of the newly liberated 5' exon forms a phosphodiester bonds with the 5' terminal P of the 3' exon, thus splicing them together.
  3. The 3' OH of the intron forms a phophodiester bond with the phosphate of the nucleotide 15 residues from the 5' end, giving a 5' terminal fragment, with the remainder of the intron cyclized.
• The 5s rRNA is processed separately and in similar fashion to the tRNAs.

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:

• How many bases are used to determine each amino acid? Obviously need at least 20 codon "words." From a simple consideration of possibilities:
  • A one base codon could code for a maximum of 4¹ = 4 amino acids, clearly not sufficient.
  • A two base codon could code for a maximum of 4² = 16 amino acids, still not enough.
  • A three base codon could code for a maximum of 4³ = 64 amino acids, which is more than adequate. Thus a three base codon is needed, but will be highly degenerate if all codons are used.
• Is the code punctuated - that is, are there signals between codons indicating the beginning and ends of codons. (For example, one combination of bases could be set aside as a "period" to indicate and set off read codons.)
• Is the code overlapping? Thus we could imagine a triplet code where every possible triplet is read such that ABCDABCD might be read as: ABC, BCD, CDA, DAB, etc. instead of ABC, DAB, etc.

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'Æ 3' corresponds to NH₂Æ 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 26-1 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.

• Of the 64 possible codons, 61 code for amino acids. The remaining three are "stop" codons (UAA = ochre, UAG = amber, and UGA = opal. The names are derived from the discoverer of UAG, Bernstein which is German for amber. The other two are puns on amber.).
• The code is very conservative, many mutations will have no effect, particularly in the third base.
• The second base determines the character of the amino acid. Thus:
  • U in the second position gives a hydrophobic amino acid.
  • C in the second position gives a neutral hydrophilic amino acid or proline.
  • G in the second position gives a basic or neutral hydrophilic amino acid.
  • A in the second position gives a hydrophilic amino acid.

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Last modified 10 April 2002