Posttranscriptional Processing of rRNA and tRNA

Both ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs)are synthesized in the form of larger transcripts (pre-rRNA and pre-tRNA, respectively), which undergo cleavage at both ends of the transcript, en route to becoming mature RNAs. The total amount of DNA encoding these RNAs amounts to less than 1% of the E. coli genome, but because of the instability of mRNA (which is encoded by the remaining 99%), rRNA and tRNA constitute about 98% of the total RNA in a bacterial cell.

rRNA Processing - The E. coli genome contains seven different operons for rRNA species. Each one encodes, sequences in a single transcript, for one copy each of 16S, 23S, and 5S rRNAs (Figure 26.41). Because the three species are used in equal amounts, the logic of this organization is apparent. Less easy to explain is that each transcript also includes sequences for one to four tRNA molecules. Because rRNAs and tRNAs are all used in protein synthesis, the interspersion of rRNA and tRNA sequences may represent a means of coordinating the rates of synthesis of these RNAs.

The initial transcript from each rRNA operon is a short-lived RNA molecule of 30S (Figure 26.41). Abnormal accumulation of this species in bacterial strains defective in RNase III first suggested a role for this enzyme in rRNA processing. In fact, one double-strand cut in each of two giant stem-loop regions releases precursors to 16S and 23S rRNAs, and the same probably occurs for 5S rRNA. Further maturation steps require the presence of particular ribosomal proteins, which begin to assemble on the precursor RNAs while transcription is still in progress. The embedded tRNA sequences are processed to give mature tRNAs, along the same routes used for other tRNA species.

tRNA Processing - Aside from the tRNAs embedded in pre-rRNA transcripts, the other tRNAs are synthesized in transcripts that contain one to seven tRNAs each, all surrounded by lengthy flanking sequences. The maturation steps are summarized in Figure 26.42, using the well-studied case of the E. coli tyrosine tRNA species (tRNA Tyr) as an example. The steps in the process can be summarized as follows:

> 1. Maturation starts with an endonuclease that cleaves at a stem - loop structure on the 3' side of the tRNA sequence.

> 2. Ribonuclease D carries out exonucleolytic cleavage to a point two nucleotides removed from the CCA sequence at the 3' end.

> 3. The 5' end is created by ribonuclease P, which cleaves to leave a phosphate on the 5' terminal G. This enzyme creates the 5' terminus of all tRNA molecules. It is not clear what structural features are recognized by RNase P, for different sequences are contained in the cleavage sites. Ribonuclease P consists of one RNA molecule of 377 nucleotides and one protein molecule with M r of about 20,000. Both components are necessary for full catalytic activity, but under nonphysiological conditions the RNA molecule alone can catalyze accurate cleavage. Thus, ribonuclease P is a ribozyme, a member of the class of catalytic RNAs.

> 4. After the proper 5' terminus has been created, ribonuclease D removes the remaining two nucleotides from the 3´ end. Should excessive "nibbling" occur through faulty control of RNase D activity, there is an enzyme that will restore the CCA end to any tRNA in a nontranscriptive fashion. This enzyme specifically recognizes the 3' terminus of tRNAs that lack the CCA end and catalyzes sequential reactions with a CTP, another CTP, and an ATP.

> 5. Creation of the modified bases common to tRNAs occurs at the final stage, including methylations, thiolations, reduction of uracil to dihydrouracil, and so forth. In the specific example of Figure 26.42, the modifications include formation of two pseudouridines, one 2-isopentenyladenosine, one O 2-methylguanosine, and one 4-thiouridine.

An additional posttranscriptional process, namely intron splicing, is almost exclusively confined to eukaryotes (see here).

See also: mRNA Turnover, Ribozymes (from Chapter 11), Structure of tRNAs (from Chapter 27), Structure of Prokaryotic mRNAs (from Chapter 27)

INTERNET LINKS:

> 1. Small RNA Database

> 2. The RNA World

> 3. RNA Modification Database

> 4. tRNA Sequence Database

> 5. rRNA Database

> 6. 5S rRNA Homepage