Human Biochemistry, Volume 2 - Murray R. 1993

Structure, Function, and Replication of Information Macromolecules
RNA Synthesis and Processing
RNA Synthesis

The process of RNA Synthesis along a DNA template is best characterized in prokaryotes. Although Gene regulation and RNA Processing in mammalian Cells differ from prokaryotic systems, the actual mechanisms of RNA synthesis are nearly identical in both types of organisms. This is why the description of prokaryotic RNA synthesis is largely applicable to Eukaryotic cells, despite differences in the Enzymes and regulatory signals involved.

The sequence of ribonucleotides in an RNA molecule is complementary to the sequence of deoxyribonucleotides in one of the DNA strands (Fig. 37.8). The specific DNA strand that directly serves as the template for transcribing RNA molecules is called the coding strand. The opposing strand is frequently referred to as the noncoding strand of the corresponding gene. It is important to note that within a double-stranded DNA molecule containing multiple genes, the coding strand for any given gene does not necessarily reside on the same physical DNA strand throughout (Fig. 39.1). In other words, a single DNA strand may function as the coding strand for some genes and as the noncoding strand for others. Note that, with the substitution of U for T, the sequence of the RNA transcript is identical to that of the noncoding strand.

DNA-dependent RNA polymerase is the enzyme responsible for polymerizing ribonucleotides into a sequence complementary to the gene's coding strand (Fig. 39.2). The enzyme binds to a specific region of the coding strand known as the promoter. Synthesis then initiates at the start point and continues until a termination sequence is reached. The region of transcribed DNA situated between the promoter and the terminator is termed the METABOLISM/31.html">Transcription unit. The resulting RNA molecule, synthesized in the 5'→3' direction, is called the primary transcript. In prokaryotic organisms, the primary transcript frequently contains RNA copies of several genes simultaneously, whereas in eukaryotes it typically represents a single gene. The 5' ends of the prokaryotic primary transcript and mature cytoplasmic RNA are identical, meaning that the transcription start point corresponds to the 5' nucleotide of the mRNA. In eukaryotes, primary transcripts synthesized by RNA polymerase II are immediately modified by The addition of a "cap"—7-methylguanosine triphosphate (Fig. 37.10)—which is consistently present at the 5' end of mature cytoplasmic mRNAs. Capping appears to be essential both for the maturation of the primary transcript and for the subsequent Introduction/27.html">Translation of the mature mRNA.

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Fig. 39.2. Polymerization of ribonucleotides into an RNA sequence complementary to the coding strand of a gene. The reaction is catalyzed by RNA polymerase. (From: J. D. Watson, Molecular Biology of the Gene, 3rd. ed., Copyright 1976, 1970, 1965, by W. A. Benjamin Inc. Menlo Park, Calif.)

The DNA-dependent RNA polymerase molecule of E. coli is composed of four subunits: two identical subunits (α subunits) and two additional subunits that are similar in size but distinct (β and β' subunits). To carry out its polymerase activity, the enzyme must form a holoenzyme—a complex comprising the core enzyme (RNA polymerase proper) and an additional protein factor (the σ factor) that promotes tighter binding of the polymerase to specific DNA promoter sequences. Bacteria produce numerous distinct σ factors, each functioning as a regulator that modulates the promoter Specificity of RNA polymerase. The appearance of different σ factors correlates temporally with the activation of distinct "complex programs" for expressing specific sets of genes in prokaryotic systems, such as bacteriophage development, sporulation, or the heat-Shock response.

The process of RNA synthesis illustrated in Fig. 39.3 involves the binding of the RNA polymerase complex to the DNA template at the promoter region. Following the initiation phase of RNA synthesis, the σ factor is released, and RNA elongation proceeds in the 5'→3' direction, antiparallel to the DNA template strand. The enzyme polymerizes ribonucleotides in a specific sequence that reflects The Structure of the coding strand via base-pairing complementarity. Pyrophosphate is released as a byproduct of this reaction. In both prokaryotic and eukaryotic organisms, RNA polymerization typically initiates with a purine ribonucleotide.

As the elongation complex—containing RNA polymerase (the core enzyme)—moves along the coding strand, the DNA must unwind to allow correct base-pairing with the incoming ribonucleotides incorporated into the growing RNA chain. The size of this unwound DNA region remains constant throughout transcription, spanning approximately 17 Base Pairs per polymerase molecule (and appears independent of the transcribed DNA sequence). This suggests that RNA polymerase is associated with an auxiliary factor possessing unwinding activity, which drives the opening of the DNA helix. The requirement for the DNA double helix to unwind and its strands to separate (at least transiently) during transcription inevitably entails some disruption of nucleosome structure.

The termination signal for RNA synthesis is a specific sequence located within the DNA coding strand. This signal is recognized by a termination protein, the ρ factor. Following the termination of an RNA chain, the core enzyme dissociates from the DNA template and, upon binding a new σ factor molecule, can recognize appropriate promoter sites to initiate the synthesis of a new RNA molecule. A single coding strand can be transcribed simultaneously by multiple RNA polymerase molecules, though the process is regulated such that at any given moment, each molecule occupies a different region of the DNA. An electron micrograph of RNA synthesis is shown in Fig. 39.4.

Several types of DNA-dependent RNA polymerases have been identified in mammalian cells, their properties summarized in Table 39.1. Each of these enzymes presumably directs the transcription of distinct gene sets. The molecular weights of the three Major Classes of mammalian RNA polymerases range between 500,000 and 600,000. Their structural Organization bears a striking resemblance to that of bacterial DNA-dependent RNA polymerase, as all contain two large subunits alongside several smaller subunits. Recent cloning and sequencing studies have demonstrated Sequence Homology between the Amino acid sequences of eukaryotic and prokaryotic RNA polymerases. The precise Functions of individual subunits remain to be fully elucidated, though some likely serve regulatory roles in recognizing specific promoter and terminator sequences.

Fig. 39.3. The process of RNA synthesis. Initiation is shown at the upper left, where the sigma factor associates with the RNA polymerase core enzyme to form a complex capable of recognizing the promoter and initiating transcription. The process concludes with the release of RNA polymerase. The free polymerase and other released catalytic factors can then participate in a new round of transcription. The abbreviation "Enz." denotes the enzyme. (From J. D. Watson, Molecular Biology of the Gene, 3rd. ed., Copyright 1976, 1970, 1965 by W. A. Benjamin Inc. Menlo Park, Calif.)

Fig. 39.4. Electron micrograph of multiple copies of transcribed ribosomal RNA genes from amphibian cells. Magnification ×6000. As shown in the photograph, the transcript length increases as RNA polymerase moves along the gene. A short transcript is associated with the near end of the gene, while a much longer one is associated with the far end. Arrows indicate the direction (5'→3') of transcription. (Reproduced with permission from Miller O. L. Jr, Beatty B. R., Portrait of a Gene. J. Cell Physiol. 1969. 74 [Suppl. 1]:225.)

Table 39.1. Nomenclature and localization of animal DNA-dependent RNA polymerases

One of the toxins, α-amanitin, produced by the mushroom Amanita phalloides, is a specific inhibitor of nucleoplasmic DNA-dependent RNA polymerase (RNA polymerase II), which has made it a valuable tool in numerous molecular biology studies (see Table 39.1).

Transcription Signals

Nucleotide sequence analysis of cloned genes has revealed A number of DNA regions that play a vital role in transcription. By studying A large number of bacterial genes, it has become possible to construct consensus models of sequences that function as transcription promoters and terminators. Bacterial promoters consist of approximately 40 nucleotide pairs (4 turns of the DNA double helix), meaning they are small enough to be completely covered by the E. coli RNA holopolymerase complex. Two short conserved elements have been identified within the consensus promoter structure. Located about 35 nucleotide pairs upstream (toward the 5' end) from the transcription start site is an octamer sequence shown in Fig. 39.5. Closer to the Transcription initiation site (at about

10 NUCLEOTIDES) lies a 6-membered AT-rich region. It has a relatively low melting Temperature due to the absence of GC pairs. Therefore, it is believed that DNA strand dissociation readily occurs in this region—known as the TATA sequence (or Pribnow box)—allowing the RNA polymerase bound to the promoter region to access the sequence of the coding strand immediately adjacent to the promoter on the 3' side.

As shown in Fig. 39.6, ρ-dependent transcription termination signals in E. coli cells are also characterized by a specific consensus structure. The conserved terminator sequence, consisting of approximately 40 nucleotides, contains spaced inverted repeats and ends with a series of AT pairs. The RNA transcript formed after the transcription complex passes through the inverted repeat region can form the intramolecular hairpin structure shown in Fig. 39.6. Transcription continues further into the aforementioned AT region, after which, under The Influence of a specific termination protein called the ρ factor, the RNA polymerase complex halts and dissociates, releasing the primary RNA transcript.

Transcription signals in mammalian genes are, as might be expected, more complex in their organization. Data obtained using Genetic Engineering indicate the presence of several types of signals that control transcription. Two Types of signal sequences are located near the promoter region itself. One of them indicates where transcription should begin, and the other determines how frequently this event should occur. In the thymidine kinase gene of the herpes virus, which uses the host transcription system to express its own genes, There is a single unique transcription initiation site. Precise transcription from this site is determined by the flanking 5' sequence located 32 to 16 nucleotides upstream from the initiation site. This region contains the sequence TATAAAAG, which is clearly homologous to the functionally related Pribnow box (TATAAT) typically found about 10 base pairs upstream from the start site of prokaryotic mRNA synthesis. RNA polymerase II likely binds to DNA in the TATA box region and initiates RNA synthesis approximately 32 nucleotides downstream—at a thymidine residue surrounded by purine nucleotides (Fig. 39.7). Thus, the TATA box is likely the precise signal that dictates where transcription should start.

Fig. 39.5. Bacterial promoters contain two highly conserved sequences located 35 and 10 nucleotides upstream (5' direction) from the transcription initiation site, designated as +1.

Fig. 39.6. Bacterial transcription termination signal, consisting of inverted repeats separated by a certain distance and an AT-rich region (top). Following transcription, this region forms the Secondary structure in the RNA transcript shown at the bottom of the figure.

Two sequence elements located further upstream from the transcription initiation site form a single functional unit that determines the frequency of transcription for a given gene. A mutation in either of these regions—located at positions —61 to —47 and —105 to —80 base pairs from the thymidine kinase gene transcription initiation site—reduces the frequency of initiation events by 10- to 20-fold. The functioning of such promoter elements, which control the accuracy and frequency of initiation, strongly depends on their position and orientation. Even a single-nucleotide substitution in this region can have a significant impact on their function. The distance to the transcription initiation site is also critical; when the 5'→3' orientation is reversed, these elements typically lose their regulatory activity (Fig. 39.8).

A third class of sequences increases or decreases the baseline level of transcription of eukaryotic genes. Depending on the effect they exert, these elements are referred to as enhancers or silencers, respectively. They can be located either upstream (5' side) or downstream (3' side) of the transcription initiation site. Unlike promoter sequences, enhancers and silencers can exert a cis effect at a distance of hundreds or thousands of bases from the corresponding transcriptional unit. Their function is orientation-independent.

Fig. 39.7. Transcription of the thymidine kinase gene. DNA-dependent RNA polymerase II binds to the region complementary to the TATA box and initiates transcription of the coding strand at a T residue surrounded by Purines, located approximately 32 nucleotides downstream from the TATA box. The first 5' purine residue in the primary transcript is rapidly modified

by the addition of a cap.

Fig. 39.8. Organization scheme of regulatory blocks in a typical eukaryotic gene. A functional gene can be divided into regulatory and structural regions separated by the transcription initiation site (indicated by an arrow). The regulatory region consists of two elements that determine the basal level of expression. The proximal element, the TATA box, directs RNA polymerase to the transcription initiation site and, consequently, determines the precision of transcription initiation. Another regulatory element (upstream) controls the frequency with which Transcription is initiated. The best-characterized regulatory element of this class is the so-called CAAT box, although other elements may be utilized in other genes. Expression regulation also involves enhancers and silencers—elements that enhance or attenuate basal transcription levels—as well as elements that regulate the expression of specific genes in response to various signals (including Hormones, heat shock, Metal Ions, and certain chemical agents). This category also includes functionally similar elements responsible for the tissue specificity of Gene Expression. It is possible that these latter two blocks of regulatory elements functionally overlap (indicated by the connecting line). The dependence of this type of element's function on orientation is indicated by arrows. For instance, the proximal element must be in the 5'→3' orientation. The CAAT box and similar elements function most efficiently in the 5'→3' orientation, although some can operate in both orientations. Dashed lines between the boxes indicate that the positions of these elements relative to the transcription initiation site are not strictly fixed. In fact, expression regulatory elements can also be located downstream (i.e., closer to the 3' end) of the transcription initiation site.

Finally, yet another class of regulatory elements is known, which provides adaptive regulation of expression for certain genes. Representatives of this class include regulatory elements sensitive to hormones (Steroids, T₃, TRH, cAMP, prolactin, etc.; see Chapter 44). This also encompasses elements that specifically regulate cellular responses to heat shock, metals (Cd²⁺ and Zn²⁺), and certain chemical toxins (dioxin). This class includes specific DNA sequence regions responsible for regulating tissue-specific gene expression, such as the Liver albumin gene. Some of these adaptive structures function similarly to silencers or enhancers (e.g., the glucocorticoid hormone-sensitive regulatory element acts as an enhancer).

A common property of all regulatory elements, both core and accessory, is that their function depends on the interaction of specific DNA regions with specific protein factors. Numerous such protein factors have been identified (Table 39.2). A significant body of research has been dedicated to studying the mechanism by which these DNA-Protein Interactions influence gene transcription.

Table 39.2. Some transcription-controlling regulatory elements and their cognate binding factors found in genes transcribed by RNA polymerase II

Transcription termination signals recognized by eukaryotic RNA polymerase II are poorly understood. However, there is evidence to suggest that termination signals are located at a considerable distance downstream of the 3' end of eukaryotic coding regions. For instance, transcription termination signals for the mouse ß-globin gene have been detected at multiple sites 1000–2000 bases past the site where transcript polyadenylation normally occurs. Little is known about the termination process itself. It remains unclear whether any specific protein factors analogous to the bacterial rho (p) factor are involved in termination. The 3' end of mature mRNA is generated after transcription is completed, seemingly in a two-step process. Once RNA polymerase II passes the region encoding the 3' end of the transcript, the primary transcript is cleaved by an RNA endonuclease approximately 15 nucleotides downstream of the AAUAAA consensus sequence. The AAUAAA sequence appears to function as an RNA Cleavage signal in eukaryotic transcripts. Subsequently, the newly formed 3' end is polyadenylated in the nucleoplasm, as described below.

DNA-dependent RNA polymerase III, which transcribes tRNA and small nuclear RNA (snRNA) genes (see Chapter 37), recognizes an intragenic promoter located entirely within the transcribed sequence. In the case of eukaryotic tRNA genes, the intragenic promoter function is carried out by two separate internal sequence blocks. These are transcribed, retained within the mature tRNA in a highly conserved region, and participate in The formation of the DHU and TΨC loops, respectively (Fig. 37.11). In vitro studies of tRNA gene structure have demonstrated that the distance between these two blocks must be 30–40 base pairs for proper promoter function. Transcription is initiated in the region between the 10th and 16th nucleotides upstream of block A. Regarding the 5S rRNA gene, which is also transcribed by RNA polymerase III, it has been shown to interact with a specific transcription protein factor. Apparently, by binding to the intragenic promoter, this factor interacts with RNA polymerase III, ensuring the precise positioning of the enzyme's catalytic center at the transcription initiation site.