transcription and post-transcriptional processing. We’ve looked at mRNA already. Here we see that rRNAs are also synthesized as pre-RNAs. But they undergo very different kinds of processing. You may recall that the genes for rRNA in eukaryotic interphase nuclei are organized into a nucleolus. This unique chromatin organization arises because of an association of proteins with not one, but hundreds of copies of rRNA genes organized one after the other as shown in this slide, in tandem. These genes are transcribed into identical pre-rRNAs that behave as 45S RNAs in sucrose density gradient centrifugation. You may know that 90% of the mass of a cell is water but most of the rest, nearly 10% is protein. So it shouldn’t come as a surprise that the cell must make lots of rRNA to maintain its stock of ribosomes. The transcription then, of tandem ribosomal DNA (or ribosomal gene copies) ensures an adequate supply of ribosomes for protein synthesis. When each 45S gene is transcribed, a 45S transcript is produced. Within each transcript is embedded 3 of the eukaryotic rRNAs: the 18S RNA, a 5.8S RNA and the 28S RNA. After transcription, the RNA between the 18S and the 5.8S and between the 5.8S and the 28S regions of the primary transcript, called transcribed spacers, are recognized and cleaved by specific rRNA endonucleases. The transcribed spacer RNA is hydrolyzed by general ribonucleases, leaving behind the mature rRNAs. In the right cells, it’s possible to visualize rRNA genes in the process of trancription. In the upper right-hand micrograph, is a frog oocyte, not yet fertilized. You may know that frogs’ eggs are quite large. During the development of these large eggs, the nucleolus undergoesselective replication. It’s a pretty unusual situation, producing many copies of nucleolar DNA without replicating other DNA in the chromosomes. So in other words, these cells have many more copies of the 45S rRNA gene than other cells in the frog. You can guess the reason for this so-called amplification. It allows this very large cell to make the unusually large number of extra ribosomes it needs for its size. It’s easy to find these genes in the electron microscope. Look at the tandem copies of the fuzzy regions along the nucleolar DNA in the electron micrograph on the right. Each of the fuzzy regions is the length expected for a 45S double-helical DNA molecule. At high magnification, each fuzzy 45S region looks like an old-fashioned ‘lampbrush’. This is a brush you might have used to clean the soot out of a a kerosene lamp chimney. Hence the name “lampbrush chromosomes”. At this magnification, one can identify likely structures. So if the region between two lampbrushes is the region between 2 45S genes, then the bristles on the 45S long lampbrush must be rRNA in the process of transcription, that is, ‘nascent’ 45S rRNAs. Note that the bristles increase in size from top to bottom in this view, which would make sense if multiple transcripts were being made from the same 45S gene, and if transcription happened to start near the top end of the lampbrush and proceeded towards the bottom. So if each bristle is a transcript, then lying along the axis of the lampbrush, as if moving down the DNA, are many RNA polymerase 1 molecules, each catalyzing the synthesis of a 45S pre-rRNA. Recall that RNA polymerase 1 is the polymerase that transcribes rRNAs. The 4th eukaryotic rRNA is a 5S molecule. There are multiple 5S rRNA genes, but they are not clustered. They are dispersed on many chromosomes, rather than being organized into a single place on one chromosome. Each 5S rRNA is transcribed by RNA polymerase 3, which has the unique property of recognizing a promoter sequence within the gene, that is within the region that will be transcribed, rather than to the left of the gene, as is the case for most promoters. After binding to this internal promoter, consisting of two short DNA sequences shown here, the polymerase shifts to the start site of transcription with the help of initiation factors, and proceeds to transcribe a 5S rRNA. Here’s how rRNA transcription and ribosome assembly are coordinated: Watch the 45S and 5S transcripts being made by their respective RNA polymerases. Next, ribosomal proteins that were already made in the cytoplasm, enter the nucleus. Some of them bind to the 45S precursor RNA. This begins assembly of both ribosomal subunits. After the two subunits have been formed, the 5S rRNA associates with the large subunit. Next, enzymatic activities that are part of the ribosomal subunits themselves, catalyze processing of the 45S transcript, the removal of the regions between the 18S, the 5.8S, and the 5.8S and the 28S rRNAs. The ribosomal subunits separate and are transported through nuclear pore complexes into the cytoplasm, where they will once again re-associate and participate in the translation of mRNAs into polypeptides. And now let’s look at tRNA genes, and their transcription and processing. tRNAs themselves are very short RNAs, less than a hundred nucleotides in length. tRNA genes are also dispersed on different chromosomes in eukaryotes, though they can often be found in clusters as shown here. tRNAs are transcribed by RNA polymerase 3, again from internal promoters just like the5S rRNA genes. Not shown here, the 5′ and 3′ ends of precursor tRNAs are trimmed from the ends. That is there are extra nucleotides at the 5′ and 3′ ends that have to be removed before the tRNA is fully functional. Also, many bases within tRNAs are chemically modified. and a tri-nucleotide, -C-C-A, is enzymatically added nucleotide by nucleotide, to the 3′ end of every tRNA molecule after transcription. The CCA trinucleotuide at the 3′ end serves as an amino acid attachment site. You may recall that tRNAs are the decoding device, with and amino acid at one end and the anticodon at the other – you can see the anticodon at the bottom of this illustartion. We’ll see more about the role of RNAs in translation, in another module, but we’re now at the end of this one.