Gene Expression Help (page 2)
In contrast to the single RNA polymerase of prokaryotes, there are three such enzymes in eukaryotes, one for each major class of RNA. The enzyme that synthesizes rRNA is RNA polymerase I (pol I). The synthesis and processing of ribosomal RNA (rRNA) occurs in one or more specialized regions of the genome called nucleoli. Multiple copies of rRNA genes in tandem array are found in each nucleolus. The promoter regions for pol I lie upstream from the start site of transcription. A Hogness box (TATA box) lies within the promoter as the eukaryote analogue of the prokaryotic Pribnowbox. The initiation of rRNA synthesis is highly species-specific; within a species, one or more proteins (essential for the transcription process) recognize promoters only in the rDNA of the same species.
RNA polymerase II (pol II) has its own specific initiation factors for synthesis of all eukaryotic mRNAs. Its promoters lie upstream from the start site of each gene, but the activity of the promoters may be increased by physically linked (i.e., in cis-position) DNA sequences called enhancers. Enhancers may function in either orientation, and may reside either within or upstream or downstream from their target genes (sometimes at great distances). The enhancing effect is mediated through sequence-specific DNA binding proteins. It is hypothesized that once the DNA binding protein attaches to the enhancer sequence, it causes the intervening nucleotides between the enhancer and the promoter to loop out and bring the enhancer into physical contact with the promoter of the gene it enhances. This loop structure then facilitates the attachment of RNA polymerase II molecules to the promoter of the transcribing gene.
The transcription termination signals for eukaryotic mRNA molecules are not known. RNA polymerase II continues elongatingm RNA chains beyond the sequences found in mature mRNAs before termination occurs by an unknown mechanism. The transcript is then somehow specifically cleaved to form the correct 3'end.
Complex mechanisms ensure that the introns are removed (spliced) from the pre-mRNA (primary transcript) and that the exons are spliced together in the proper order. Thereafter, the pre-mRNAs of eukaryotes undergo a number of covalent modifications before they are released from the nucleus as mature messenger molecules. The enzyme poly-A polymerase adds (without a template) a long stretch of adenine nucleotides to the 3' end of each pre-mRNA, forming a poly-A tail. Since only mRNA molecules (not rRNAs or tRNAs) have these tails, it might be suggested that they have something to do with translation. In contrast to most mRNAs, however, those for histone proteins inmost species do not acquire poly-A tails. So the function of these tails remains amystery. The 5' ends become "capped" with an unusual guanine nucleotide (3'-G-5'ppp5'-N-3'p). A methyl group is subsequently added to this backward guanine cap. Thus, both the 5' and 3' ends of most eukaryotic mRNAs possess free 2'- and 3'-OH groups on their terminal ribose sugars. Bacterial mRNAs contain specific ribosome binding sites in their leader sequences; eukaryotic mRNAs do not have these sites. Instead, a eukaryotic ribosome usually binds to the mRNA cap and then moves downstream along the mRNA until it encounters the first AUG initiation codon, and begins translation there.
Eukaryotic ribosomes, like their bacterial counterparts, consist of two major subunits, but they aremore complex, existing as 40S and 60S subunits that together form an 80S complex. The rRNA components in more complex eukaryotes (e.g., angiosperms, vertebrates), having sedimentation coefficients of 18S, 5.8S, and 28S, are transcribed from 50 to 5000 identical genes tandemly arranged in that order into massive clusters located on one or more chromosomes as nucleolar organizing regions (NORs).When active, these rRNA repeat units extend out from the main chromosome fiber as elongated threads (see Problem 13.23).When complexed with specific proteins involved in rRNA synthesis and processing, these clusters become visible under the light microscope as nucleoli, where the assembly of ribosomes begins. The number of NORs per haploid genome varies with the species from one to several. In E. coli, there are only seven copies of the rRNA genes. Very few bacterial genes exist in multiple copies, and even when they do the copy number is very small. As much as half of the eukaryotic primary rRNA transcript may be lost during processing of themature rRNA molecule. Some of this loss is due to the removal of introns. In the protozoan ciliate Tetrahymena thermophila, in vitro rRNA transcripts appear to be self-splicing.
The eukaryotic genes encoding tRNAs generally also exist in multiple copies, from 10 to several hundred for each tRNA species per haploid genome. The identical genes within each tRNA family tend to be widely dispersed in species with relatively low numbers of tRNA gene copies. In organisms with more highly reiterated tRNA genes, they may form heteroclusters containing several kinds of tRNA genes. RNA polymerase III (pol III) is responsible for synthesizing not only all of the tRNAs but also 5S ribosomal RNA and other small RNAs. These transcripts are usually short (less than 300 nucleotides), with complementary end sequences that may allow formation of a stable base-paired stem. Sequences within the tRNA genes are required for transcription by pol III. Internal control regions (located inside the genes themselves) also direct termination of transcription by pol III. Thus, the same region can function both biosynthetically (in the gene) and structurally (in the RNA product).
The process of translating an mRNA into a polypeptide chain in eukaryotes is essentially the same as that in bacteria, but differs in several important ways. Whereas only three well-defined initiation factors are required for translation of E. coli mRNAs, many more are needed in eukaryotes. Eukaryotic initiation factors are designated eIFs to distinguish them from their bacterial counterparts.
EXAMPLE 13.2 A tRNAMet (symbolized Met-tRNAMet when activated) brings an unformylated methionine into the first position on the ribosome. Hydrolysis of ATP to ADP is required for mRNA binding. The 40S ribosomal subunit is then thought to attach to the mRNA at its capped 5' terminus, and then it slides along (consuming ATP) until it reaches the first AUG codon. Normally, only AUG is an efficient initiator codon in eukaryotes, whereas UUG, GUG, and AUU may also be used in E. coli.
EXAMPLE 13.3 Three different elongation factors (EFs) in eukaryotes replace those found in bacteria. However, a single termination factor (RF) replaces RF1 and RF2 of bacteria. RF recognizes all three stop codons (UAC, UAA, and UGA).
A nascent polypeptide chain may not become biologically active until after it has been modified in one or more specific ways, such as being enzymatically phosphorylated, glycosylated, or partly digested by peptidase enzymes. Phosphorylation involves the addition of one or more phosphate groups (Example 13.4) and glycosylation involves the addition of one or more carbohydrate groups to the protein sequence. Peptidase enzymes cleave the protein into smaller units (Example 13.5).
EXAMPLE 13.4 Protein kinases are enzymes that transfer terminal phosphate groups from ATP to specific amino acids on target proteins. Phosphorylation of these proteins may either raise or lower their biological activities. For example, the skeletal muscle enzyme glycogen synthetase is inactivated after phosphorylation, whereas phosphorylation of the enzyme glycogen phosphorylase increases its activity.
EXAMPLE 13.5 The hormone insulin is synthesized as a single-chain precursor (proinsulin) with little or no hormonal activity. Two internal cuts remove 31 amino acids from pro-insulin, producing the two polypeptide chains of the functional dimer that are held together by disulfide bonds. Likewise, human growth hormone that circulates in blood is a ''clipped'' version of the pituitary form of that hormone.
Practice problems for these concepts can be found at:
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