Chapter 2 Section 4 NUCLEIC ACIDS
2.4.3. TYPES OF RNA
rewind it. Topoisomerases relieve tortional stress that develops in cellular DNA molecules during replication or other processes. They hydrolyze a phosphodiester linkage in one strand of the double helix, relax the supercoiling by rotating one strand around the other, and then reseal the break. DNA gyrases induces negative supercoiling in relaxed, closed-circular DNA.
DNA segment could theoretically yield two mRNAs with different sequences and thus, different protein -coding capabilities. However, normally only one strand of a DNA duplex gives rise to useful information when transcribed into mRNA. The non-coding complementary, strand of DNA is termed anti-sense. Anti-sense mRNA is an RNA complementary in sequence to one or more mRNAs. In some organisms, the presence of an anti-sense mRNA can inhibit gene expression by base-pairing with the specific mRNAs. In molecular biology research, this effect has been used to study gene function, by simply shutting down the studied gene by adding its anti-sense mRNA transcript. Such studies have been done on the worm Caenorhabditis elegans and the bacterium Escherichia coli. This plays a part in RNA interference.
The prokaryotic and eukaryotic mRNAs differ in their requirement for post-transcriptional processing (see Figure 2.4.14 below). Since the prokaryotic system has no nucleus and the genes are closely packed with no non-coding intervening sections, the mRNA formed can be translated from the 5’ end even as the 3’ end is still being copied. Prokaryotic mRNA is thus essentially mature upon transcription and requires no processing, except in rare cases. However, in eukaryotes the genes, even those devoted to a single pathway, are physically separated in the DNA and may even be on different chromosomes. Moreover, the functional mRNA with a continuous protein-coding sequence is much smaller than the primary transcript since eukaryotic genes exist in pieces of coding sequences called exons, separated by non-coding segments, the introns. Thus the primary mRNA transcript has to be broken to clip away the non-coding sections and then carefully stitched back to yield the functional mRNA. The short-lived, unprocessed mRNA is known as pre-mRNA and when completely processed it is termed mature mRNA.
These post-transcriptional modifications, collectively called RNA processing, therefore, are a prerequisite for mRNA formation and its translation. Consequently, mRNA transcription and translation cannot proceed simultaneously as it does in prokaryotes.
Fig. 2.4.14. The transfer of information from DNA to protein. The transfer proceeds by means of an RNA intermediate called messenger RNA (mRNA). In procaryotic cells the process is simpler than in eukaryotic cells. In
Pre mRNA Processing
The first step in processing is the addition of a cap to the initiating (5’) nucleotide of the primary transcript. The 5' cap consists of a terminal 7-methylguanosine residue which is linked through a 5'-5'-triphosphate bond to the first transcribed nucleotide. This modification is critical for recognition and proper attachment of mRNA to the ribosome, as well as protection from 5' exonucleases. It may also be important for other essential processes, such as splicing and transport. This modification occurs even before transcription is complete, so the 5’ cap is present in the primary transcript.
Processing at the 3’ end entails the generation of a free 3’-hydroxylgroup to which a string of adenylic acid residues is added by the enzyme poly(A) polymerase. This process, called polyadenylation, aids in mRNA stability by protecting it from exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation.
The final step in processing is known as splicing (Fig. 2.4.14). This is the process by which pre- mRNA is modified to remove the stretches of non-coding sequences called introns; the stretches that remain include protein-coding sequences and are called exons. Sometimes pre-mRNA messages may be spliced in several different ways, allowing a single gene to encode multiple proteins. This process is called alternative splicing. Splicing is usually performed by an RNA- protein complex called the splicesome, but some RNA molecules are also capable of catalyzing their own splicing.
Once processing is completed, the mature mRNA is exported through the nuclear pores to the cytoplasm.
Transfer RNA
The major role of tRNA is to translate mRNA sequence into amino acid sequence.The nucleotide sequence of mRNA is converted into the amino acid sequence of protein with the help of two types of adaptor molecules, tRNAs and enzymes called aminoacyl-tRNA synthetases.
Transfer RNAs are the smallest of the three kinds of RNA. Several different species of tRNA can be found in every cell as there is at least one tRNA that bonds specifically to each of the 20 amino acids commonly occurring in proteins. Since the genetic code is “degenerate” and there are 61 possible codons which specify amino acids, and since there are only 20 amino acids, one amino acid may have more than one codon. Leucine, serine and arginine, for example, are each specified by six different codons. The number of tRNAs in most cells is more than the number of amino acids found in proteins (20) and is also more than the number of codons in the genetic code (61).
Thus many amino acids have more than one tRNA to which they can attach (which explains how there can be more tRNAs than amino acids).
The functioning of tRNA molecules, which are 70 – 80 nucleotides long, depends on their three- dimensional structure. Some nucleotides in tRNA have been modified, such as dihydrouridine, pseudouridine, and inosine. In dihydrouridine, a hydrogen atom is added to each C5 and C6 of uracil. In pseudouridine, the ribose is attached to C5, instead of the normal N1. Inosine plays an important role in codon recognition. In addition to these modifications, a few nucleosides are methylated.
All tRNA molecules resemble a cloverleaf with stem-loop arrangements (Figure 2.4.15). The four stems are short double helices stabilized by Watson-Crick base pairing; three of the four stems
have loops containing seven or eight bases at their ends, while the remaining, unlooped stem contains the free 3’ and 5’ ends of the chain.
Fig. 2.4.15. A general diagram for the structure of tRNA. The positions of invariant bases as well as bases that seldom vary are shown in color. The numbering system is based on yeast tRNAPhe. R= purine; Y = pyrimidine.
Dotted lines denote sites in the D loop and variable loop regions where varying numbers of nucleotides are found in different tRNAs.(Source: Garrett, R. H. and Grisham, C.M., 1999, p 386, fig. 12.34).
The double-helical regions form as hairpin turns and bring complementary stretches of bases in the chain into contact. Because of the arrangement of the complementary stretches along the chain, the overall pattern of H-bonding appears as a cloverleaf. Each cloverleaf consists of four H-bonded segments—three loops and the stem where the 3’- and 5’-ends of the molecule meet.
These four segments are designated the acceptor stem, the D loop, the anticodon loop, and the TΨC loop.
Three nucleotides, termed the anticodon, located at the center of one of the loops, can form base pairs with the three complementary nucleotides forming the codon in mRNA. Specific aminoacyl- tRNA synthetases recognize each tRNA for a specific amino acid and covalently attach the proper amino acid to the unlooped amino acid acceptor stem. The 3’ end of all tRNAs has the sequence CCA, which is in most cases added after synthesis and processing of the tRNA are complete.
Many cells contain fewer than 61 tRNAs and thus there can be more codons in a cell than tRNAs.
This means that many tRNAs can attach to more than one codon. This would not be possible if perfect Watson-Crick base pairing were demanded between codons and anticodons. Codon- anticodon base pairing is somewhat less stringent than the standard A-U and G-C base pairing.
Therefore, a single tRNA anticodon can recognize more than one codon corresponding to a given amino acid. This broader recognition can occur because of non-standard pairing between bases in
G·C pair. Thus, a given anticodon in tRNA with G in the first (wobble) position can base-pair with the two corresponding codons that have either pyrimidine (C or U) in the third position. (Figure 2.4.16). For example, the phenylalanine codons UUU and UUC (5’→3’) are both recognized by the tRNA that has GAA (5’→3’) as the anticodon. In fact, any two codons of the type NNPyr (N
= any base; Pyr = pyrimidine) encode a single amino acid and are decoded by a single tRNA with G in the first (wobble) position of the anticodon.
Although adenine rarely is found in the anticodon wobble position, many tRNAs in plants and animals contain inosine (I), a deaminated product of adenine, at this position. Inosine can form nonstandard base pairs with A, C, and U. A tRNA with inosine in the wobble position thus can recognize the corresponding mRNA codons with A, C, or U in the third (wobble) position (see Figure 2.4.16). For this reason, inosine-containing tRNAs are heavily employed in translation of the synonymous codons that specify a single amino acid. For example, four of the six codons for leucine (CUA, CUC, CUU, and UUA) are all recognized by the same tRNA with the anticodon 3’-GAI-5’; the inosine in the wobble position forms nonstandard base pairs with the third base in the four codons. In the case of the UUA codon, a nonstandard G·U pair also forms between position 3 of the anticodon and position 1 of the codon.
Fig. 2.4.16. Nonstandard codon-anticodon base pairing at the wobble position. The base in the third (or wobble) position of an mRNA codon often forms a nonstandard base pair with the base in the first (or wobble) position of a tRNA anticodon. Wobble pairing allows a tRNA to recognize more than one mRNA codon (top); conversely, it allows a codon to be recognized by more than one kind of tRNA (bottom), although each tRNA will bear the same amino acid. Note that a tRNA with I (inosine) in the wobble position can “read” (become paired with) three different codons, and a tRNA with G or U in the wobble position can read two codons. Although A is theoretically possible in the wobble position of the anticodon, it is almost never found in nature.
(Source: Lodish et al, 2003. p 123, fig. 4.23).
Thus the standard wobble pairings make it possible to fit the 20 amino acids to 61 codons with as few as 31 kinds of tRNA molecules; in animal mitochondria a more extreme wobble allows
Ribosomal RNA
Ribosomal RNAs are constituents of the protein synthesizing machinery of the cell, the ribosome.
Ribosomes, are composed of two subunits, called small and large, and ribosomal RNAs are integral components of these subunits. The functional ribosome essentially provides a scaffold for the binding of mRNA, transfer RNA (tRNA), and a number of protein factors involved in promoting the different steps of protein synthesis. The ribosome has a complex architecture comprising three or more ribosomal RNA (rRNA) molecules and more than 50 protein molecules.
The ribosome is approximately globular, its average diameter ranging from 2.5nm (Escherichia coli) to 2.8nm (mammalian). The small subunit comprises one RNA and between 21 (E. coli) and 42 (rat liver) proteins, while the large subunit comprises two or three RNAs, with the number of proteins ranging from 34 (E. coli) to 46 (rat liver).
The ribosomal RNAs (rRNAs) lie at the core of the protein synthesis machinery. These RNAs were long regarded as mere scaffolds for the ribosomal proteins (r-proteins) but recent work has shown that the rRNAs in fact carry out the key reactions in translation. A major function of the ribosomal proteins is ensuring the correct structure of the rRNA, allowing its tight packing around the active centre of the ribosome.
The RNA component of the ribosome accounts for about 60% to 65% of the total weight. To study the properties and functions of the ribosomes, scientists have resorted to dissociation of the ribosomes into their components by lowering the Mg2+ concentration of the medium and then studying their reassociation with an increase in the Mg2+ concentration using the technique of analytical ultracentrifugation.
In contrast to tRNAs, rRNA molecules are fairly large and considerably fewer types exist in the cell. They are intimately associated, both structurally and functionally, with the proteins in the ribosome. As mentioned earlier, in all organisms the ribosome consists of two subunits. These are designated the 40S and 60S subunits in eukaryotes and the 30S and 50S subunit in Bacteria, Archaea and the cytoplasmic organelles of eukaryotes, mitochondria and chloroplasts. In almost all organisms the small ribosomal subunit contains a single RNA species (the 18S rRNA in eukaryotes and the 16S rRNA elsewhere). In Bacteria and Archaea, the large subunit contains two rRNA species (the 5S and 23S rRNAs); in most eukaryotes the large subunit contains three RNA species (the 5S, 5.8S and 25S/28S rRNAs).
The smallest RNA component of the large subunit, present in almost all types of ribosome, is 5 S rRNA. It is highly conserved among all kingdoms of life. For many years 5 S rRNA was used as a model molecule for studies on RNA structure and RNA - protein interactions, and as a
in a number of structural studies and comparative sequence analyses. In Eukaryota and Archaea, the structure of 5 S rRNA is better preserved than in Eubacteria, where much more nucleotide sequence variability is observed. In spite of extensive research, the precise role of 5S rRNA is not yet fully determined. One of its functions may be to maintain large-subunit stability.
Detailed information of the folding of rRNAs in the ribosome has emerged from landmark crystallographic studies of intact ribosomes and ribosomal subunits of organisms such as Haloarcula marismortui and Thermus thermophilus. These studies have shown that there is a remarkable conservation of the secondary structures of ribosomal RNAs in organisms. This conservation is a reflection of the fact that ribosomes are ancient RNA machines whose function (and, hence structure) is conserved in all living cells.
Fig. 2.4.17. Secondary structure of human 5 S rRNA. In all organisms, the structure consists of five double- stranded regions (I - V) and five loops (A - E). Loop E, which differs between eukaryotic/archaeal and eubacterial 5 S rRNAs, is highlighted in yellow. The palindromic motifs within eubacterial loop E are boxed.
Ribosomal RNA Processing
As in the case of the eukaryotic mRNAs, the rRNAs in almost all organisms are not synthesized as simple transcripts, but are generated from large precursors called pre-rRNAs, by posttranscriptional processing. In bacteria, 16S, 23S, and 5S rRNAs arise from a single 30S RNA precursor of about 6,500 nucleotides. RNA at both ends of the 30S precursor and segments between the rRNAs are removed during processing. The endonuclease RNAase III cleaves stem structures formed by complementary sequences that flank each of the mature rRNA sequences.
The separated pre-rRNAs are 3’ processed by the 3’ to 5’ exoribonuclease RNAase T and 5’
processed by the endonuclease RNAase E. RNAase III also processes other RNA substrates, messenger RNAs (mRNAs) and phage and plasmid transcripts, while RNAase T participates in the processing of tRNAs and other stable RNAs.
Pre-rRNA processing is less well understood in eukaryotes. In particular, many of the processing enzymes remain to be identified. Processing is posttranscriptional, with the exception of the initial cleavage by RNAase III, which, at least in yeast, is cotranscriptional. Subsequent processing shows a strong 5’ – 3’ bias in the order of cleavage. In yeast, processing of the 18S rRNA involves four endonuclease cleavages, but the endonucleases responsible have not been identified.
In both yeast and E. coli (the two organisms in which RNA processing is best understood) RNA- processing enzymes are not specific to a single pathway, but have a range of RNA substrates.
The rRNA is synthesized in the nucleolus. These machines then self-assemble into the two complex folded structures (the large and the small subunits) in the presence of 70 – 80 ribosomal proteins. The function of the rRNA is to provide a mechanism for decoding mRNA into amino acids (at the center of small ribosomal subunit) and to interact with the tRNAs during translation by providing peptidyltransferase activity (large subunit). Accuracy of translation is provided by both subunits.