Lippincott's Illustrated Reviews Cell and Molecular Biology PDF

Transcription I. OVERVIEW Transcription refers to the first step in gene expression, the copying of a particular sequence of deoxyribonucleic acid (DNA) into messenger ribonucleic acid (mRNA). Genes are considered expressed when the information contained within D...

Transcription I. OVERVIEW Transcription refers to the first step in gene expression, the copying of a particular sequence of deoxyribonucleic acid (DNA) into messenger ribonucleic acid (mRNA). Genes are considered expressed when the information contained within DNA has been converted to proteins that affect cellular properties and activities. The DNA-directed synthesis of mRNA is a necessary intermediate to produce protein. In order for an mRNA to be produced, a gene sequence on DNA needs to be identified, along with information necessary for the exact start site. Genes are split into exons and introns, and the entire region is initially transcribed. This primary RNA transcript is processed before it exits the nucleus. Once created, mRNA is modified through RNA splicing, 5' end capping, and the addition of a poly(A) tail after which the mature mRNA enters the cytoplasm. Generally, every gene contains two classes of information, one to specify the primary structure of the final product and the other to regulate the expression of the gene. Both the timing and the amount of RNA pro- duced are regulated during transcription. mRNA encodes the amino acid sequence of proteins, and both ribosomal and transfer RNAs directly par- ticipate in protein synthesis. II. TYPES OF RNA Several distinct types of RNA are known: ribosomal (rRNA), transfer (tRNA), messenger (mRNA), and other small noncoding RNA, each with its own distinctive structure and function. These RNAs (tRNA and rRNA) are transcribed but not translated and are considered noncoding RNAs (ncRNAs). There are additional small RNAs that are also noncoding, like the ones in the nucleolus (snoRNAs) nucleus (snRNA) and cytoplasm (miRNA) that perform specialized functions. A. Ribosomal RNA rRNA accounts for approximately 80% of total RNA in the cell and asso- ciates with proteins to form ribosomes. Eukaryotes have several differ- ent rRNA molecules: 18S, 28S, 5S, 5.8S, 18S, and 28S. Ribosomes are important during protein synthesis as they contain peptidyltrans- ferase "activity; an activity catalyzed by ribozymes (Figure 8.1 ). 92 II. Types of RNA 93 Promoter Promoter Promoter Promoter DNA Transcription mRNA Figure 8.1 Different types of eukaryotic RNA. B. Transfer RNA tRNA is the smallest of the three RNAs. It functions in the protein syn- thesis by virtue of its ability to carry the appropriate amino acid and also provide a mechanism by which nucleotide information can be translated to amino acid information through its anticodon. C. Messenger RNA mRNA carries genetic information from DNA to cytosol for translation. About 5% of the total RNA within a cell is mRNA.It is the most hetero- geneous in terms of size and carries specific information necessary for the synthesis of different proteins. D. MicroRNA miRNAs, like the other RNA molecules, are encoded by genes and are single-stranded RNA molecules about 21 to 23 nucleotides in length. These newly discovered molecules are transcribed but not translated. They function in regulating gene expression by their ability to bind mRNA and to down-regulate the gene expression. Distinct enzymes catalyze the synthesis of various RNAs as shown in Table 8.1. Table 8.1: Eukaryotic RNA Polymerases Polymerase RNA Products * RNA polymerase I Ribosomal RNAs mana RNA polymerase II Messenger RNA, microRNA, and some noncoding RNAs RNA polymerase Ill Transfer RNAs 94 8. Transcription Poly-A addition site 5'end "upstream" Figure 8.2 Structure of a typical eukaryotic gene. Ill. GENE STRUCTURE AND REGULATORY ELEMENTS IN EUKARYOTIC PROTEIN-CODING GENES The minimal linear sequence of genomic nucleic acids that encode pro- teins and structural RNA is termed a gene (Figure 8.2). Gene sequences are written from 5' (5 prime) to 3'. Eukaryotic genes are composed of coding exons, noncoding introns, and noncoding consensus sequences. The number of introns and exons, their size, location, and sequence dif- fer from gene to gene. Noncoding regions at the 5' end to the first exon are referred to as upstream sequences and those at the 3' end are called downstream sequences. A. Consensus sequences Consensus sequences are evolutionarily conserved and act as rec- ognition markers and define a potential DNA recognition site. They are usually bound by proteins (transcription factors) and other regu- latory proteins that recognize a particular sequence. 1. Promoters: Promoters are DNA sequences that select or deter- mine the start site of RNA synthesis. The consensus sequence for promoters typically has the sequence ''TATA" (or variations ofT and A) and is often located 15 to 30 base pairs (bp) upstream from the transcription start site, called a TATA box and an initiator sequence (lnr) near the RNA start site at+ 1 (Figure 8.3). Additional sequences that may be required for promoter function include the CMT box -130 -120 -110 -40 -30 -20 -10 + +10 -Base position Upstream elements Downstream Figure 8.3 Promoter elements found upstream to the coding sequences in a gene. IV. RNA Synthesis 95 and the GC box. In eukaryotes, proteins known as transcription or basal factors bind to the TATA box and facilitate the binding of RNA polymerase II. 5'end 3'end 2. Splice acceptor and donor sequences: Splice acceptor and donor sequences are one type of consensus sequence found at 3' splice the 5' and 3' ends of introns.lntrons nearly always begin with gua- acceptor site nine and uracil (GU) nucleotides and end with adenine and gua- nine (AG) nucleotides, which are preceded by a pyrimidine-rich tract (Figure 8.4). This particular consensus sequence is essential for splicing introns out of the primary transcript. Figure 8.4 Splice acceptor and donor sequences. IV. RNA SYNTHESIS Synthesis of RNA from DNA occurs in the nucleus and is catalyzed by an RNA polymerase. RNA differs significantly from DNA in that it is single stranded and contains uracil (U) instead of the thymine (T) found in DNA. Protein-encoding genes produce mRNA as an intermediate to the cyto- sol for protein synthesis. Regulatory mRNA sequences are important for stability and translational efficiency. These are sequences in the 5' and the 3' ends of the mRNA, called untranslated regions (UTR), and are not part of the final protein product. A. RNA polymerases There are several distinct RNA polymerases in eukaryotic cells as shown in Table 8.1. The mechanism described below refers to RNA polymerase II, which catalyzes the synthesis of mRNA from protein- coding genes. B. Several proteins bind to the gene to be transcribed The reaction catalyzed by RNA polymerase II requires the formation of a large complex of proteins over the start site of the gene. This preinitiation complex is important for accurately positioning the RNA polymerase II on DNA for initiation. This complex consists of general transcription factors and accessory factors. C. Regulatory regions An mANA-producing eukaryotic gene can be divided into its coding and regulatory regions as defined by the transcription start site. The cod- ing region contains the DNA sequence that is transcribed into mRNA, which is ultimately translated into a protein. The regulatory region con- sists of two classes of sequences (Figure 8.5). One class is responsible for ensuring basal expression and the other for regulated expression. 1. Basal promoters: Basal promoter sequences generally have two components. The proximal component, generally the TATA box, directs RNA polymerase II to the correct site and a distal compo- nent specifies the frequency of initiation (CAAT and GC boxes). The best studied of these is the CAAT box, but several other sequences may be used in various genes. These sequences determine how frequently the transcription event occurs. Mutations in these regions reduce the frequency of transcriptional starts 96 8. Transcription Other Proximal..... regulatory element, elements TATAbox.. wiL-__::::.......Iillll..a. Regulated expression Basal expression Upstream elements Downstream Figure 8.5 Two types of regulatory sequences. 10 to 20 fold. Typical of these DNA sequences are the GC and CAAT boxes, so named because of the DNA sequences involved. These boxes bind specific proteins and the frequency of transcrip- tion initiation is a consequence of these protein-DNA interactions, whereas the protein-DNA interaction at the TATA box ensures fidelity of initiation. 2. Enhancers and response elements: Enhancers and response elements regulate gene expression. This class consists of sequences that enhance or repress expression and of others that mediate the response to various signals including hormones, chem- icals, etc. Depending upon whether they increase or decrease the initiation rate of transcription, they are called enhancers or repres- sors and have been found both upstream and downstream from the transcription start site. In contrast to proximal and upstream promoter sequences, enhancers and repressors can exert their effects even when located hundreds or even thousands of bases away from the transcription units located on the same chromo- some. They also function in an orientation-independent fashion. These regions are bound by proteins (specific transcription factors) that regulate gene expression and are discussed in Chapter 10. D. Basal transcription complex formation Basal transcription requires, in addition to RNA polymerase II, a number of transcription factors called A, B, D, E, F, and H, some of which are composed of several different subunits (Figure 8.6). These general transcription factors are conventionally abbreviated as TFII A, B, etc. (transcription factor, class II gene). TFIID (consists of TATA binding protein [TBP] + 8 to 10 TBP-associated factors), which binds to the TATA box, is the only one of these factors capable of bind- ing to specific sequences of DNA. Binding of TBP to the TATA box in the minor groove causes a bend in the DNA helix. This bending is thought to facilitate the interaction of TBP-associated proteins with other components of the transcription initiation complex and, possi- bly, with other factors bound to the upstream sequences. One of the transcription factors, TFIIF, has DNA helicase activity that promotes the unwinding of the DNA near the transcription start site. This allows the opening of the complex to allow for transcription. RNA polymerase II is also phosphorylated in its C-terminal domain, which allows it to extricate from the promoter and begin elongating a transcript. IV. RNA Synthesis 97 Enhancer Initiators of activation assemble to activate RNA polymerase. RNA polymerase progresses along DNA template leaving complex behind. Initiation complex dissipates upon departure of RNA polymerase. Figure 8.6 Formation of the transcription complex requires several proteins in addition to RNA polymerase II. 98 8. Transcription E. A single-stranded RNA is produced from a double-stranded DNA Eukaryotic RNA polymerase is a DNA-dependent RNA polymerase as it uses information from DNA to synthesize a complementary sequence. Only one strand of the gene is used as a template for tran- scription and is referred to as the template strand. The product is a complementary single-stranded RNA. RNA polymerase reads DNA 3' to 5' and produces an RNA molecule complementary to it (see Figure 8.6}. Clinical Application 8.1: Bacterial DNA-Directed RNA Synthesis is Inhibited by the Antibiotic Rifampin Rifampin specifically inhibits bacterial RNA synthesis by interfering with the bacterial RNA polymerase. The inhibited enzyme remains bound to the promoter, thereby blocking the initiation by uninhibited enzyme. Rifampin is especially useful in the treatment of tuberculosis. This drug along with iso- niazid (an antimetabolite) has greatly reduced morbidity due to tuberculosis. Clinical Application 8.2: Retroviruses, Such as Human Immunodeficiency Virus (HIV), have an RNA Genome Retroviruses, such as HIV and human T-cell lymphotropic virus, contain reverse transcriptase, an enzyme that copies the RNA genome of the virus into a eDNA. "Reverse" signifies that the biological information flows from RNA to DNA, opposite the usual direction of transfer. Reverse transcrip- tase mediates the RNA template-dependent information of double-stranded DNA from a single-stranded RNA by an intricate process. The transcribed DNA is integrated into the host cellular genome and is replicated with the host cellular machinery. Clinical Application 8.3: AZT and DDI Inhibit Reverse Transcriptase of HIV Many useful antiviral drugs act as anti metabolites because they are structur- ally similar to pyrimidine or purine bases. Drugs, such as zidovudine (AZT) and ddl (dideoxyinosine), undergo phosphorylation by host cellular kinases to form nucleotide analogues, which are incorporated into the viral nucleic acids resulting in chain termination. Selective toxicity results because viral enzymes are more sensitive to inhibition by these antimetabolites than mammalian polymerases. V. RNA PROCESSING REACTIONS Gene transcription produces an RNA that is larger than the mRNA found in the cytoplasm for translation. This larger RNA, called the primary tran- script or heterogeneous nuclear RNA (hnRNA}, contains segments of transcribed introns. The intron segments are removed and the exons are V. RNA Processing Reactions 99 joined at specific sites, called donor and acceptor sequences, to form the Cytoplasm mature mRNA by a mechanism of RNA processing (Figure 8.7). Lipid bilayer A. Addition of a 5' cap Almost immediately after the initiation of RNA synthesis, the 5' end of RNA is capped by a methyl guanosine residue, which protects it ///I Nucleus from degradation (by 5' exonucleases that digest DNA from a free 5' mRN7 l J or 3' end) during elongation of the RNA chain. The cap also helps the transcript bind to the ribosome during protein synthesis. B. Addition of a poly(A) tail The primary transcripts contain a highly conserved AAUAAA consen- sus sequence, known as a polyadenylation signal, near their 3' end. The polyadenylation site is recognized by a specific endonuclease that cleaves the RNA approximately 20 nucleotides downstream. Transcription may proceed for several hundred nucleotides beyond Nucleoplasm the polyadenylation site, but the 3' end of the transcript is discarded. Lipid The newly created 3' terminus, however, serves as a primer for enzy- bilayer matic addition by poly(A) polymerase of up to 250 adenine nucle- Cytoplasm otides (Figure 8.8). The poly A tail also serves as a mechanism of protection of the mRNA from degradation (Chapter 10). C. lntron removal Figure 8.7 Splice sites are present within the gene and delineate the introns. mRNA is transcribed and processed in Splice site sequences, which indicate the beginning (GU) and end- the nucleus. ing (AG) of each intron, are found within the primary RNA transcript. lntrons are removed and exons are spliced Gained) together to form the mature mRNA (Figure 8.9). A special structure called the spli- ceosome converts the primary transcript into mRNA. Spliceosomes comprise the primary transcript, five small nuclear RNAs (U1, U2, US, and U4/6), and more than 50 proteins. Collectively called snRNPs (pronounced "snurps"), the complex facilitates this process by posi- tioning the RNA for necessary splicing reactions and helps form the structures and intermediates for removal of the intron. The mature RNA molecule now leaves the nucleus by passing into the cytoplasm through the pores in the nuclear membrane. Clinical Application 8.4: Mutations in Splicing 5' ' cap Signals Cause Human Disease Thalassemias are hereditary anemias that comprise the single most com- mon genetic disorder in the world. The mutations that cause the thalassemia 5' affect the synthesis of either the alpha or the beta chains of globin, causing a decreased production of hemoglobin and, consequently, an anemia. Point mutations can occur within the TATA box or mutations can occur in the splice junction sequences at the intron-exon boundaries. Some of the splicing abnormalities alter the sequence GT at the beginning 5' cap of an intron or the AG at the end. Because these sequences are absolutely required for normal splicing, such mutations lead to the loss of beta-globin production. In the case of other mutations that affect the consensus region of the donor or acceptor site, there is a reduced ability of the RNA to correctly splice and it will result in decreased but detectable amounts of beta-globin. Figure 8.8 RNA processing reactions. 100 8. Transcription Clinical Application 8.5: Transcription-Coupled Repair TFIIH, a general transcription factor involved in transcription of all genes, also plays a role in nucleotide excision repair in eukaryotic cells. Some of Spliceosome: small the subunits have homology to helicases, which aid in the unwinding of nuclear ribonucleo- DNA at the start site during transcription. This sharing of subunits between protein particles transcription and repair processes might explain why there is efficient repair (snRNPs) combined with primary transcript. of actively transcribed regions more than nontranscribed regions (transcrip- tion-coupled repait}. In this system when there is distortion in DNA, and Exon I RNA polymerase is unable to transcribe due to this hindrance, a complex of proteins known as CSA and CSB is recruited to the site. These proteins aid in the opening of the double-stranded DNA and recruitment of the general transcription factor TFIIH, which allow in the opening and subse- quent removal of the affected region. CSA and CSB derive their name from Cockayne syndrome, a rare inherited disorder where these proteins are Exon I defective due to mutations. Spliced exons from mature mRNA. Mature mRNA Figure 8.9 mRNA splicing. Chapter Summary RNA polymerase II transcribes protein-coding genes. Transcription requires several factors to bind to the regulatory region of the gene. Proximal and distal promoters and other regulatory sequences control gene expression. RNA is transcribed in the nucleus and undergoes processing before entering the cytoplasm. RNA processing reactions include addition of a 5' methyl guanosine cap, a poly(A) tail, and splicing of introns out of the heterogeneous nuclear transcript. Study Questions Choose the ONE best answer. 8.1 What will be the sequence of the single-stranded RNA transcribed from the following segment of double- Correct answer = B. RNA polymerase reads double- stranded DNA? stranded DNA on the template strand (running 3' to 5') and synthesizes a complementary single-stranded RNA 5'-TTGCACCTA-3' molecule. RNA contains uracil in place of thymine. So the 3' -AACGTGGAT-5' sequence of the newly synthesized RNA would be similar to A. 5'-UAGGUGCUU-3' the sequence on the coding strand, except in places where B. 5'-UUGCACCUA-3' thymine occurs. C. 5'-AACGUGGUA-3' D. 5'-AUCCACGUU-3' E. 5'-UUCGUGGAU-3' Study Questions 101 8.2 Addition of a 5' 7-methyl guanosine cap to the primary RNA transcript during nuclear processing Correct answer= C. A 5' 7-methyl guanosine group is added to the newly synthesized RNA, and it helps protect the RNA A. Facilitates the assembly of the spliceosome complex. from degradation by enzymes in the cell. The spliceosome B. Identifies the transcript as a transfer RNA molecule. complex is assembled around the intron-exon boundary C. Protects the RNA against degradation by cellular during splicing. Unlike mRNA, tRNA molecules are not mod- exonucleases. ified. The presence of the cap on the mRNA is also impor- D. Inhibits translation of the RNA molecule into protein. tant for ribosomes to bind to the mRNA during translation. E. Prevents RNA molecules from forming double- stranded complexes. 8.3 Splicing of a newly synthesized RNA molecule to remove introns and join exons Correct answer= B. Spliceosomes are complexes made up of small nuclear RNA and proteins involved in the process A. Occurs in the rough endoplasmic reticulum of the of removal of introns and splicing of exons. The processing cytosol. reactions take place in the nucleus of the cell, and transla- B. Involves a complex of small nuclear RNA and tion occurs in the cytosol. Rifampin inhibits the initiation of protein molecules. transcription, and bacterial RNA is not processed similar to C. Proceeds concurrently with translation. eukaryotic RNA. The rate of transcription is stimulated by transcription factors. D. Is inhibited in bacteria by the drug rifampin. E. Is stimulated by the binding of transcription factors to the RNA. 8.4 Which of the following is an mRNA processing reaction? Correct answer= D. RNA processing consists of removal of A. Binding of the RNA polymerase to TATA box intron sequences from the newly produced heterogeneous B. Synthesis of an RNA strand using RNA polymerase I nuclear RNA. Transcription is initiated by the formation of C. Addition of 7-methyl guanosine residues to the 3' a preinitiation complex. Addition of 7-methyl guanosine end of the mRNA occurs in the 5' end of the mRNA. D. Removal of introns from the heterogeneous nuclear RNA E. None of the above 8.5 Which of the following sites on a gene is important for recognition of the beginning and the ends of intron Correct answer = C. GT and AG are sequences recognized sequences? at the beginning and the ends of introns and are important during splicing of exons. TATA, GC, and CAAT boxes are A. TATAbox promoter sequences, and a poly(A) tail is added to the 3' B. GCbox end of the mRNA as part of the processing reaction. c. Splice sites GT and AG D. Poly(A) tail E. CAATbox