S

Processing

18S rRNA

28S rRNA

Many transcription factors are involved in controlling gene expression in eukaryotes.

RNA polymerase I transcribes the genes for the two large ribosomal RNA molecules and RNA polymerase III transcribes the genes for tRNA, 5S rRNA and a few other small RNA molecules. RNA polymerase II transcribes most eukaryotic genes that encode proteins and as a result is subject to the most complex regulation. Since ribosomal RNA and transfer RNA are needed all the time by all types of cells, RNA polymerases I and III operate constitutively in most cell types.

A variety of proteins, known as transcription factors are also needed for the correct functioning of RNA polymerases. Transcription factors may be divided into general transcription factors and specific transcription factors. General transcription factors are needed for the transcription of all genes transcribed by a particular RNA polymerase, and are typically designated TFI, TFII, TFIII followed by individual letters. The I, II, and III refer to the corresponding RNA polymerase (see below). Specific transcription factors are needed for transcription of particular specific gene(s) under specific circumstances. [Proteins such as the sigma subunit of bacterial RNA polymerase may also be regarded as transcription factors, however, this terminology is usually only used for eukaryotes.]

Eukaryotes contain many copies of the genes for ribosomal RNA. These are found in clusters and are transcribed by RNA polymerase I.

Transcription of rRNA and tRNA in Eukaryotes

The genes for the two large ribosomal RNAs are present in multiple copies, from seven in E. coli to several hundred in higher eukaryotes (Fig. 6.16). In bacteria, the copies are dispersed, but in eukaryotes they form clusters of tandem repeats. In humans, there are clusters of rRNA genes on five separate chromosomes. The 18S and the 28S rRNA are transcribed together as a single large RNA (45S RNA) that is cleaved to release the two separate ribosomal RNA molecules. Between these transcription units are non-transcribed spacer regions. In eukaryotic cells, the rRNA genes have their own RNA polymerase to transcribe them, RNA polymerase I.

Synthesis of rRNA is localized to a special zone of the nucleus known as the nucle-olus. Here the rRNA precursor is both transcribed and processed into 18S and 28S rRNA. These rRNA molecules then bind proteins, giving ribonucleo-protein particles. This yields a dense granular region when seen under the microscope (Fig. 6.17). The segments of chromosomes associated with the nucleolus were named "nucleolar organizers." It is now known that these correspond to the clusters of rRNA genes.

nucleolar organizer Chromosomal region associated with the nucleolus;actually a cluster of rRNA genes RNA polymerase I Eukaryotic RNA polymerase that transcribes the genes for the large ribosomal RNAs RNA polymerase II Eukaryotic RNA polymerase that transcribes the genes encoding proteins

RNA polymerase III Eukaryotic RNA polymerase that transcribes the genes for 5S ribosomal RNA and transfer RNA transcription factor Protein that regulates gene expression by binding to DNA in the control region of the gene

Transcription of rRNA and tRNA in Eukaryotes 147

FIGURE 6.17 Ribosomal RNA is Made in the Nucleolus

A) Electron micrograph of a thin-sectioned nucleolus from a mouse cell fixed in situ. Black arrows indicate peri-nucleolar condensed chromatin and the asterisk shows dense fibrillar components (d) clumping around fibrillar centers (f). Granular regions (g) of newly made ribonucleoproteins are also marked. Image provided by Ulrich Scheer, University of W├╝rzburg. B) Spread Christmas tree structure (4 microns long) from a mouse cell is shown at the same magnification as (A). Bar represents 0.5 micron. From: Raska I., Oldies but goldies: searching for Christmas trees within the nucleolar architecture. Trends in Cell Biology 13 (2003) 517-525.

Transcription of rRNA and tRNA in Eukaryotes 147

RNA polymerase III transcribes genes for small non-coding RNAs, in particular tRNA and 5S rRNA.

Although most promoters are AT rich, presumably because the weaker base pairs help in opening up the DNA, the promoter for RNA polymerase I is unusual in containing many GC pairs. There are two GC-rich regions, the core promoter and the upstream control element, that are 80 to 90 percent identical in sequence (Fig. 6.18). Both are recognized by protein UBF1 (Upstream Binding Factor 1), a single polypeptide. After UBF1 has bound, another protein, selectivity factor SL1, binds next to it. SL1 consists of four polypeptides, one of which, TBP (TATA Binding Protein), is also required for RNA polymerases II and III (see below). Once UBF1 and SL1 are in place, RNA polymerase I can bind. It is uncertain how the binding of UBF1 and SL1 at the upstream control element helps initiation in the case of RNA polymerase I. However, in similar cases, the DNA is known to bend around, bringing the upstream element into direct contact with the promoter region.

RNA polymerase III is responsible for making 5S rRNA and transfer RNA. It also makes some small nuclear RNAs, while other snRNAs are transcribed by RNA polymerase II (see below). The promoters for 5S rRNA and tRNA are unique and somewhat bizarre in being internal to the genes. Transcription of these genes requires the binding of either of two proteins known as TFIIIA and TFIIIC to a region over 50 bp downstream from the start site (Fig. 6.19). Once these have bound, they enable TFIIIB to bind to the region around the start of transcription. TFIIIB consists of three polypeptides, including TBP, and positions RNA polymerase III correctly at the start site.

As the promoters for RNA polymerase I and RNA polymerase III illustrate, recognition factor sites may be upstream or downstream from the start of transcription. However, in both cases, a positioning factor (SL1 or TFIIIB, respectively) is required to make sure that the polymerase starts transcribing at the correct place. These positioning factors thus play a similar role to that of the sigma factor in bacteria.

FIGURE 6.18 RNA Polymerase I Transcribes rRNA Genes

The promoter for RNA polymerase I has an upstream control element and a core promoter, the latter rich in GC sequences. The UBF1 protein recognizes and binds to both the upstream control element and the core promoter. Subsequently, SL1 binds to the DNA in association with UBF1. Finally, RNA polymerase I binds and transcription commences. How this binding pattern facilitates transcription of rRNA is not known.

FIGURE 6.19 Internal Promoter for RNA Polymerase III

The gene for 5S rRNA is transcribed using a promoter located within the gene itself. The recognition sites are downstream of the start site. TFIIIC (or TFIIIA) binds to both sites and this induces TFIIIB to bind to the promoter near the start site. Only after TFIIIB binds can RNA poly merase III bind.

FIGURE 6.19 Internal Promoter for RNA Polymerase III

The gene for 5S rRNA is transcribed using a promoter located within the gene itself. The recognition sites are downstream of the start site. TFIIIC (or TFIIIA) binds to both sites and this induces TFIIIB to bind to the promoter near the start site. Only after TFIIIB binds can RNA poly merase III bind.

RNA polymerase II transcribes genes that code for proteins.

Transcription of Protein-Encoding Genes in Eukaryotes

RNA polymerase II transcribes most eukaryotic genes that encode proteins. Recognition of the promoter and initiation of transcription by RNA polymerase II requires a number of general transcription factors. In addition, since many protein-encoding genes vary markedly in expression, a variety of specific transcription factors are needed for expression of certain genes under particular circumstances. For example, in a multi-

Transcription of Protein-Encoding Genes in Eukaryotes 149

Enhancer region

Promoter region

Gene

Transcription

Transcription w/wmmw/m

100 bp

FIGURE 6.20 Promoter and Enhancer

Although one RNA polymerase is used to transcribe most protein encoding genes, specificity is controlled by transcription factors and their recognition sequences. The promoter region is close to the start site and usually binds several transcription factors. In addition, extra transcription factors bind to regions known as enhancers. These may be far upstream of the promoter, as shown, or may be located downstream. Binding of the transcription factors to their recognition sequences influences polymerase activity and gene expression.

100 bp

Some transcription factors bind to the promoter region, others to distant enhancer sequences.

The TATA box is the critical sequence that allows RNA polymerase II to recognize the promoter.

cellular organism, different cell types produce different types of proteins. Thus, red blood cells produce hemoglobin, whereas white blood cells make antibodies. Further, protein production often varies during development. Fetal hemoglobin is different from the adult version.

The assorted transcription factors bind to and recognize specific sequences on the DNA. These DNA sequences are of two major classes, those comprising the promoter itself and a variety of enhancer sequences (Fig. 6.20).The general transcription factors for RNA polymerase II (TFII factors) bind to the promoter region. However, although some of the specific transcription factors also bind to the promoter region, others bind to the enhancer.

In eukaryotes, many protein-encoding genes are interrupted by introns. These are removed at the RNA stage. Consequently, transcription of DNA to give RNA does not yield messenger RNA directly. The RNA that results from transcription is known as the primary transcript and must be processed as described in Chapter 12 to give mRNA. The present discussion will therefore be limited to the transcription of genes by RNA polymerase II to give the primary transcript.

Promoters for RNA polymerase II consist of three regions, the initiator box, the TATA box and a variety of upstream elements (see below). The initiator box is a sequence found at the site where transcription starts. The first transcribed base of the mRNA is usually A with a pyrimidine on each side, as in bacteria. The consensus is weak: YYCAYYYYY (where Y is any pyrimidine).About 25 base pairs upstream from this is the TATA box, an AT-rich sequence, which is recognized by the same factor TBP (TATA binding protein or TATA box factor) that is needed for binding of RNA poly-merases I and III. TBP is unusual in binding in the minor groove of DNA. (Almost all DNA-binding proteins bind in the major groove). On both sides of the TATA box are GC-rich regions (Fig. 6.21).

TBP is found in three different protein complexes, depending on whether RNA polymerase I, II or III is involved. In the present case, TBP forms part of a transcription factor complex known as TFIID that is needed to recognize promoters specific for RNA polymerase II. Several other TFII complexes are also needed for RNA polymerase II function. TFIIA and TFIIB bind next. Then at last RNA polymerase II itself enhancer Regulatory sequence outside, and often far away from, the promoter region that binds transcription factors initiator box Sequence at the start of transcription of a eukaryotic gene primary transcript RNA molecule produced by transcription before it has been processed in any way TATA binding protein (TBP) Transcription factor that recognizes the TATA box

TATA box Binding site for a transcription factor that guides RNA polymerase II to the promoter in eukaryotes TATA box factor Another name for TATA binding protein upstream element DNA sequence upstream of the TATA box in eukaryotic promoters that is recognized by specific proteins

Dna Upstream Upstream TATA Initiator Gene

_element element box box ^m

Promoter

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