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The First Page of Teaching Plan No. course Biochemistry specialty Clinic medicine class 2015-2 lecturer Yan Chen period 8 students’ level undergraduate professiona l title Biochemistry associate professor time of writing 2016.11 chapter RNA Biological Synthesis----Transcription time of using 2016-2017(1) objectives and requiremen ts 1. The template, enzymes and processing of RNA biological synthesis. (First class) 2. Post-transcriptional modification in eukaryotes. (Second class) Keys:Template keys and difficulties and RNA polymerase;processing of RNA biological synthesis; Post-transcriptional modification in eukaryotes(mRNA) Difficulties: initiation, and termination in prokaryotes updated informatio n no review the content last class(20min); the template and enzymes (80min); processing of RNA arrangeme nt biological synthesis(100min); Post-transcriptional modification in eukaryotes(50min). ribozyme(20min); Reverse Transcription(20min); The processes differ in DNA and RNA synthesis(35min); discuss and summarize(35min). teaching methods Using CAI to explain, enlightening method Lippincott’s illustrated review :Biochemistry Pamela C. Champe wilkins 2009 books and references teachers’ group discussion about the plan Biochemistry the second edition High Education Press 2002 author: Reginald H. Garrett, Charles M. Grisham According to learn the RNA polymerase, then to emphasized the classification and its function of RNA polymerase in eukaryotes. The processing of RNA biological synthesis in prokaryotes should be lectured clearly. Agreement. comments from the department Lippincott’s willam & Sign name: (Content) Lesson plan for page Chapter 10 RNA Biosynthesis ----Transcription I.Teaching Goals It is based on a mastery of transcription in prokaryotes, then to be familiar with RNA processing in eukaryotes. II.Teaching Demands 1.Master the template, enzymes and transcription process in prokaryotes. 2.Familiar with RNA processing in eukaryotes. 3.Understand initiation and termination of transcription in eukaryotes. III.Teaching Contents 1. The concept of transcription and the concept of RNA replication 2.Template and enzymes The template for RNA synthesis and RNA polymerase subunits of prokaryotes. The function of RNA polymerase subunits in prokaryotes. Classification and function of RNA polymerase in eukaryotes. The inhibitor of RNA polymerase. 3.The process of transcription Three phases of transcription: initiation, elongation and termination in prokaryotes. The transcription of initiation and termination in eukaryotes (self-study). 4.Posttranscriptional processing of RNAs in eukaryotes The posttranscriptional processing of mRNA, tRNA and rRNA. IV. Class Hour 8 hours (Content) Lesson plan for page Chapter 10 RNA synthesis REVIEW According to what has been called the central dogma of molecular genetics, the function of DNA is to store information and pass it on to RNA, while the function of RNA is to read, decode and use the information received from DNA to make proteins. Reverse Transcription Three fundamental processes take place in the transfer and use of genetic information: Replication is the process by which a replica, or identical copy, of DNA is made. Replication occurs every time a cell divides so that information can be preserved and handed down to offspring. This is similar to making a copy of a file onto a disk so you can take that file to a different computer. Transcription is the process by which the genetic messages contained in DNA are "read" or transcribed. The product of transcription, known as messenger RNA (mRNA), leaves the cell nucleus and carries the message to the sites of protein synthesis. (This tutorial explains later why this step is necessary in organisms with a nucleus!) Translation is the process by which the genetic messages carried by mRNA are decoded and used to build proteins. What is RNA? RNA is structurally similar to DNA. Both nucleic acids are sugar-phosphate polymers and both have nitrogen bases attached to the sugars of the backbone- but there are several important differences. 1.They differ in composition: (1)The sugar in RNA is ribose, not the deoxyribose in DNA (as we previously learned). (2)The base uracil is present in RNA instead of thymine. 2.They also differ in size and structure: (1)RNA molecules are smaller (shorter) than DNA molecules. (2)RNA is single-stranded, not double-stranded like DNA. Lesson plan for page (Content) 3.Another difference between RNA and DNA is in function. DNA has only one function-STORING GENETIC INFORMATION in its sequence of nucleotide bases. But there are three main kinds of ribonucleic acid, each of which has a specific job to do. Ribosomal RNAs-exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes. Ribosomes are small, granular structures where protein synthesis takes place.Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% protein. Messenger RNAs-are the nucleic acids that "record" information from DNA in the cell nucleus and carry it to the ribosomes and are known as messenger RNAs (mRNA). Transfer RNAs-The function of transfer RNAs (tRNA) is to deliver amino acids one by one to protein chains growing at ribosomes. Part I Template and DNA-directed RNA polymerase 1.Template The process of converting the information contained in a DNA segment into proteins begins with the synthesis of mRNA molecules containing anywhere from several hundred to several thousand ribonucleotides, depending on the size of the protein to be made. Each of the 100,000 or so proteins in the human body is synthesized from a different mRNA that has been transcribed from a specific gene on DNA. One question which you must ask yourself is: "Why do we need mRNA if DNA holds all the genetic information, the instructions for the proteins the cell is supposed to produce?" The answer for eukaryotic cells (those cells with a nucleus) is the importance of DNA. If DNA is damaged in any way, then the coding sequence is changed and a mutation could result which could greatly affect the cell or even the whole organism! You'll learn more about this when we discuss mutations in class. Because of this, the DNA should be protected as much as possible. If the DNA were to venture out into the cytoplasm where the ribosomes are in order to give the instructions for which proteins were to be made, then it would be more vulnerable to damage from: chemicals ,UV light , or other agents. This presents a problem, however... How is the DNA supposed to get the information it encodes out to the ribosomes which carry out the instructions in the cytoplasm? The answer is that there must be a MESSENGER. This messenger is mRNA! So, how is mRNA made? Lesson plan for page (Content) Messenger RNA is synthesized in the cell nucleus by transcription of DNA, a process similar to DNA replication. As in replication, a small section of the DNA double helix unwinds, and the bases on the two strands are exposed. RNA nucleotides (ribonucleotides) line up in the proper order by hydrogen-bonding to their complementary bases on DNA, the nucleotides are joined together by a DNA dependent RNA polymerase enzyme, and mRNA results. UNLIKE what happens in DNA replication where both strands are copied, only ONE of the two DNA strands is transcribed into mRNA (remember that RNA is a single-stranded molecule). Asymmetric transcription:The two complementary DNA strands have different roles in transcription. The strand that serves as template for RNA synthesis is called the template strand(also known as the sense strand). The DNA strand complementary to the template, the nontemplate strand, or coding strand (also called the informational or antisense strand). Since the template strand and the coding strand are complementary, and since the template strand and the mRNA molecule are also complementary, it follows that the messenger RNA molecule produced during transcription is a copy of the DNA coding strand! It is called this because, with the exception of T for U changes, it corresponds exactly to the sequence of the primary transcript, which encodes the protein product of the gene. 2.DNA-directed RNA polymerase All RNA polymerases are dependent upon a DNA template in order to synthesize RNA. Both RNA and DNA polymerases can add nucleotides to an existing strand, extending its length. However, there is a major difference between the two classes of enzymes: RNA polymerases can initiate a new strand but DNA polymerases cannot. Classes of RNA Polymerases In prokaryotic cells, all 3 RNA classes are synthesized by a single polymerase. E. coli has a single DNA-directed RNA polymerase that synthesizes all types of RNA. E. coli RNA polymerase is composed of five subunits: twoαsubunits, and one for eachβ,β', and σ subunit.β(151 kD) andβ' (156 kD) are significantly larger than α(37 kD). The two α subunits are essential for assembly of the enzyme and activation by some regulatory proteins. β’,functions in DNA binding,β binds the nucleoside triphosphate substrates and interacts with σ. The σ subunit is also known as the σ factor. It binds transiently to the core and directs the enzyme to specific initiation sites on the DNA (described below). Several different forms of σ subunits have been identified, with molecular weights ranging from 28 kD to 70 kD. These five subunits(α2ββ'σ) constitute the RNA polymerase holoenzyme. Nucleotide synthesis is carried out by four subunits(α2ββ') which together are called the core polymerase. In this case, the holoenzyme includes the core polymerase and the σ factor. Lesson plan for page (Content) In eukaryotic cells there are 3 distinct classes of RNA polymerase, RNA polymerase (pol) I, II and III. However, the eukaryotic RNA polymerase does not contain any subunit similar to the E. coli σ factor. Therefore, in eukaryotes, transcriptional initiation should be mediated by other proteins. (The capacity of the various polymerases to synthesize different RNAs was shown with the toxin-amanitin. At low concentrations of toxin-amanitin synthesis of mRNAs are affected but not rRNAs nor tRNAs. At high concentrations, both mRNAs and tRNAs are affected. These observations have allowed the identification of which polymerase synthesizes which class of RNAs.) Each polymerase is responsible for the synthesis of a different class of RNA. RNA pol I is responsible for rRNA synthesis (excluding the 5S rRNA).There are 4 major rRNAs in eukaryotic cells designated by there sedimentation size. The 28S, 5S 5.8S RNAs are associated with the large ribosomal subunit and the 18S rRNA is associated with the small ribosomal subunit. RNA pol II synthesizes the mRNAs and some of the small nuclear RNAs (snRNAs) involved in RNA splicing. It is undoubtedly the most important among the three classes of RNA polymerases. RNA pol III synthesizes the tRNAs, the 5S rRNA and some snRNAs. But how do the polymerase know where to begin? In other words, where does one gene start and stop and the next one begin? The starting point of a gene is marked by a certain base sequence which is called a promoter site. Analysis and comparison of sequences in many different bacterial promoters have revealed similarities in two short sequences located about 10 and 35 base pairs away from the point where RNA synthesis is initiated.By convention the base pair that begins an RNA molecule is given the number +1, so these sequences are commonly called the -10 and -35 regions. The most common nucleotides form what is called a consensus sequence.For most promoters in E. coli and related bacteria, the consensus sequence for the -10 region also called the Pribnow box is TATAAT, and the consensus sequence at the -35 region is TTGACA. These sites are recognized by a factor called "σ". It is sigma's job to recognize the promoter sites and "tell" the DNA dependent RNA polymerase where to begin transcription. Once the RNA polymerase has been directed to the start point of the gene by σ, the σ factor is released and the RNA polymerase carries out the process of transcription. Lesson plan for page (Content) Part II Processes of Transcription 1.Prokaryotic Transcription 1.1 Initiation Unlike DNA polymerase, RNA polymerase does not require a primer to initiate synthesis. Initiation of RNA synthesis, however, occurs only at specific sequences called promoters (described last). RNA synthesis usually starts with a GTP or ATP residue, whose5'triphosphate group is not cleaved to release PP, form 5'pppGpN-OH3'; and remains intact throughout transcription. Since the role of σ factor is mainly to initiate transcription, it will be released after first phosphodiester bonds have been polymerized. 1.2 Elongation This enzyme, DNA-directed RNA polymerase, requires, in addition to a DNA template, all four ribonucleoside 5'-triphosphates (ATP, GTP, UTP, and CTP) as precursors of the nucleotide units of RNA, as well as Mg2+. The purified enzyme also contains Zn2+. The fundamental chemistry of RNA synthesis has much in common with DNA synthesis. RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3'-hydroxyl end of the RNA chain and thus builds RNA chains in the 5'→3' direction. The 3'-hydroxyl group acts as nucleophile, attacking at the a-phosphate of the incoming ribonucleoside triphosphate and releasing pyrophosphate. The overall reaction is (NMP)n + NTP (NMP)n+1 + PPi RNA polymerase requires DNA for activity and is most active with a double-stranded DNA as template.Only one of the two DNA strands is used as a template, copied in the 3'→5' direction (antiparallel to the new RNA strand) just as in DNA replication. Each nucleotide in the newly formed RNA is selected by Watson-Crick base-pairing interactions; uridylate (U) residues are inserted in the RNA opposite to adenylate residues in the DNA template, adenylate residues are inserted opposite to thymidylate residues. Guanylate and cytidylate residues in DNA specify cytidylate and guanylate, respectively, in the new RNA strand. During transcription the new RNA strand base-pairs temporarily with the DNA template to form a short length of hybrid RNA-DNA double helix, which is essential to the correct readout of the DNA strand. The RNA in this hybrid duplex "peels off '' shortly after its formation. To enable RNA polymerase to synthesize an RNA strand complementary to one of the DNA strands, the DNA duplex must unwind over a short distance, forming a transcription "bubble." Lesson plan for page (Content) (Because the DNA is a helix, this process requires considerable rotation of the nucleic acid molecules. Rotation is restricted in most DNAs by DNA-binding proteins and other structural barriers, and a moving RNA polymerase generates waves of positive supercoils ahead of and negative supercoils behind the point at which transcription is occurring. This transcription-driven supercoiling of DNA has been observed both in vitro and, in bacteria, in vivo. In the cell, the topological problems caused by transcription are relieved through the action of topoisomerases.) Once begun, transcription in E. coli proceeds at a rate of about 50-100 nucleotides per second. Elongation of the RNA strand continues until the core polymerase reaches the termination site. 1.3 Termination Following termination the core polymerase dissociates from the template. The core and sigma subunit can then reassociate forming the holoenzyme again ready to initiate another round of transcription. In E. coli transcriptional termination occurs by both factor-dependent and factor-independent means. Two structural features of all E. coli factor-independently terminating genes have been identified. One feature is the presence of new segments that are capable of forming a stem-loop structure in the RNA and the second is a downstream A rich sequence in the template. The formation of the stem-loop in the RNA destabilizes the association between polymerase and the DNA template. This is further destabilized by the weaker nature of the AU base pairs that are formed, between the template and the RNA, following the stem-loop. This leads to dissociation of polymerase and termination of transcription. Most genes in E. coli terminate by this method. The promoter sites act as a "start sign". Similarly, there are other base sequences at the end of a gene that signal a to mRNA synthesis. Just as there is a sigma factor to help signal the beginning of a gene, another factor called "rho " aids in terminating the process of transcription. Factor-dependent termination requires the recognition of termination sequences by the termination protein, rho. When the end of the gene is near, the rho factor binds to the mRNA (that's right, the mRNA, NOT the DNA) and interacts with the RNA polymerase. The interaction of rho with the RNA polymerase causes the enzyme to "fall off" the DNA template strand, thus stopping transcription! Lesson plan for page (Content) 2. Eukaryotic Transcription Eukaryotic transcription is more complex than prokaryotic transcription and, until recently, it has seemed that every eukaryotic gene was unique requiring its own transcription machinery. However, it is now possible to simplify the story somewhat. The promoters for different genes are different. Each contains a combination of sites to which specific protein factors bind. All of these factors help RNA polymerase to bind in the correct place and to initiate transcription. However, the repertoire of transcription factors and transcription factor binding sites is not unlimited. Transcription exhibits several features that are distinct from replication: 1. Transcription initiates, both in prokaryotes and eukaryotes, from many more sites than replication. 2. There are many more molecules of RNA polymerase per cell than DNA polymerase. 3. RNA polymerase proceeds at a rate much slower than DNA polymerase (approximately 50-100 bases/sec for RNA versus near 1000 bases/sec for DNA). 4. Finally the fidelity of RNA polymerization is much lower than DNA. This is allowable since the aberrant RNA molecules can simply be turned over and new correct molecules made. 5. Transcription does not require a primer, replication need. Part III Posttranscriptional Processing of RNAs When transcription of bacterial rRNAs and tRNAs is completed they are immediately ready for use in translation. No additional processing takes place. Translation of bacterial mRNAs can begin even before transcription is completed due to the lack of the nuclear-cytoplasmic separation that exists in eukaryotes. The ability to initiate translation of prokaryotic RNAs while transcription is still in progress affords a unique opportunity for regulating the transcription of certain genes. An additional feature of bacterial mRNAs is that most are polycistronic. This means that multiple polypeptides can be synthesized from a single primary transcript. Polycistronic mRNAs are very rare in eukaryotic cells but have been identified. The mitochondrial genomes in mammals and the slime mold, Dictyostelium discoideum, encode polycistronic mRNAs that are processed into primarily mono-, di-, and tricistronic transcripts. In addition, several viruses encode polycistronic RNAs. In contrast to bacterial transcripts, eukaryotic RNAs (all 3 classes) undergo significant post-transcriptional processing. All 3 classes of RNA are transcribed from genes that contain introns. Lesson plan for page (Content) The sequences encoded by the intronic DNA must be removed from the primary transcript prior to the RNAs being biologically active. The process of intron removal is called RNA splicing. Additional processing occurs to mRNAs. The 5' end of all eukaryotic mRNAs are capped with a unique 5'→5' linkage to a 7-methylguanosine residue. The capped end of the mRNA is thus, protected from exonucleases and more importantly is recognized by specific proteins of the translational machinery. 1. 5'-Cap of Eukaryotic mRNA Structure of the 5'-Cap of Eukaryotic mRNAs 2. 3' poly A tail Messenger RNAs also are polyadenylated at the 3' end. A specific sequence, AAUAAA, is recognized by the endonuclease activity of by polyadenylate polymerase which cleaves the primary transcript approximately 11 - 30 bases 3' of the sequence element. A stretch of 20 - 250 A residues is then added to the 3' end by the polyadenylate polymerase activity. The poly(A) tail also is bound by a specific protein. It is likely that poly(A) tail and their associated proteins help protect the mRNA from enzymatic destruction. 3. modification of tRNA and rRNA In addition to intron removal in tRNAs, extra nucleotides at both the 5' and 3' ends are cleaved, the sequence 5'-CCA-3' is added to the 3' end of all tRNAs and several nucleotides undergo modification. There have been more than 60 different modified bases identified in tRNAs. Both prokaryotic and eukaryotic rRNAs are synthesized as long precursors termed preribosomal RNAs. In eukaryotes a 45S preribosomal RNA serves as the precursor for the 18S, 28S and 5.8S rRNAs. The S number given each type of rRNA reflects the rate at which the molecules sediment in the ultracentrifuge. The larger the number, the larger the molecule(but not proportionally). Lesson plan for page (Content) 4. Splicing of RNAs A primary transcript for a eukaryotic mRNA typically contains sequences encompassing one gene. The sequences encoding the polypeptide, however, usually are not contiguous. Instead, in the majority of cases, the coding sequence is interrupted by noncoding tracts called introns; the coding segments are called exons. In a process called splicing, the introns are removed from the primary transcript and the exons joined to form a contiguous sequence specifying a functional polypeptide. The 2 most common are the group I and group II introns. Many of the group I and group II introns are self-splicing, i.e. no additional protein factors are necessary for the intron to be accurately and efficiently spliced out. The third class of introns is also the largest class found in nuclear mRNAs. This class of introns undergoes a splicing reaction similar to group II introns in that an internal lariat structure is formed. However, the splicing is catalyzed by specialized RNA-protein complexes called small nuclear ribonucleoprotein particles (snRNPs, pronounced snurps). Part Ⅳ Reverse Transcription and ribozyme After the RNA retrovirus enters a host cell, its genomic RNA will be transcribed into a double stranded DNA and then integrated into the host DNA. The RNA to DNA transcription is called reverse transcription. The entire process is catalyzed by reverse transcriptase which has both DNA polymerase and RNase H activities. RNA-directed DNA polymerases, also called reverse transcriptases. These enzymes transcribe the viral RNA into DNA. This process can be used experimentally to form complementary DNA. Many eukaryotic transposons are related to retroviruses, and their mechanism of transposition includes an RNA intermediate. RNA-directed RNA polymerases, or replicases, are found in bacterial cells infected with certain RNA viruses. They are template-specific for the viral RNA. The existence of catalytic RNAs and pathways for the interconversion of RNA and DNA has led to speculation that the earliest living things were made up entirely or largely of RNA molecules that served both for information storage and for catalysis of replication. The self splicing introns and the RNA component of RNase P (the enzyme that cleaves the 5' end of tRNA precursors) form a new class of biological catalysts called ribozymes. These have the properties of true enzymes and are effective catalysts. They promote two types of reaction, hydrolytic cleavage and transesterification, using RNA as substrate. Lesson plan for page (Content) Summary Transcription is very similar to replication in terms of chemical mechanism, polarity (direction of synthesis), and use of a template. The two processes differ, however, in that transcription does not require a primer, it generally involves only short segments of a DNA molecule, and within those segments only one of the two DNA strands serves as a template. Replication and transcription differ in one important respect. During replication the entire chromosome is copied to yield daughter DNAs identical to the parent DNA, whereas transcription is selective: only particular genes or groups of genes are transcribed at any one time. The transcription of DNA can therefore be regulated so that only genetic information needed by the cell at a particular moment is transcribed. Specific regulatory sequences indicate the beginning and end of the segments of DNA to be transcribed, as well as which DNA strand is to be used as template. Transcription is catalyzed by DNA-directed RNA polymerase, a complex enzyme that synthesizes RNA complementary to a segment of one strand (the template strand) of duplex DNA, starting from ribonucleoside 5'-triphosphates. To initiate transcription, RNA polymerase binds to a DNA site called a promoter. Bacterial RNA polymerase requires a special subunit for recognizing the promoter. As the first committed step in transcription, binding of RNA polymerase to promoters is subject to many forms of regulation.In E. coli transcriptional termination occurs by both factor-dependent and factor-independent means. Eukaryotic cells have three different types of RNA polymerases. Transcription stops at specific sequences called terminators. Many copies of an RNA chain can be transcribed simultaneously from a single gene. Ribosomal RNAs and transfer RNAs are made from longer precursor RNAs that are trimmed by nucleases, and some bases are modified enzymatically to yield the mature RNAs. In eukaryotes, messenger RNAs are also formed from longer precursors. Primary RNA transcripts often contain noncoding regions called introns, which are removed by splicing. Messenger RNAs are also modified by addition of a 7-methylguanosine residue at the 5' end, and cleavage and polyadenylation at the 3' end to form a long poly(A) tail.Many bases in tRNAs are also modified, mature tRNAs are replete with unusual bases not found in other nucleic acids. (Content) Lesson plan for page The processes of DNA and RNA synthesis are similar in some points. (1) The general steps of initiation, elongation, and termination with 5’to 3’ polarity (2) Large, multicomponent intiation cinokexes; These processes differ in several important ways, including the following: DNA RNA Precursors dNTP NTP Polymerase DNA Polymerase RNA Polymerase Base-pairing rules Product A-T, C-G two-strands DNA T-A, A-U, C-G mRNA, tRNA, rRNA two strand asymmetric transcription Template (1) Ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides; (2)Adherence to Wastson-Crick base-pairing rules. But U replaces T as the complementary base pair for A in RNA; (3) Only a very small portion of the genome is transcribed into RNA, whereas the entire genome must becopied during DNA replication; (4) There is no proofreading function during RNA transcription. (5) A primer is not involved in RNA synthesis. Lesson plan for page (Content) Select reading Clinical Significances of Alternative and Aberrant Splicing The presence of introns in eukaryotic genes would appear to be an extreme waste of cellular energy when considering the number of nucleotides incorporated into the primary transcript only to be removed later as well as the energy utilized in the synthesis of the splicing machinery. However, the presence of introns can protect the genetic makeup of an organism from genetic damage by outside influences such as chemical or radiation. An additionally important function of introns is to allow alternative splicing to occur, thereby, increasing the genetic diversity of the genome without increasing the overall number of genes. By altering the pattern of exons, from a single primary transcript, that are spliced together different proteins can arise from the processed mRNA from a single gene. Alternative splicing can occur either at specific developmental stages or in different cell types. This process of alternative splicing has been identified to occur in the primary transcripts from at least 40 different genes. Depending upon the site of transcription, the calcitonin gene yields an RNA that synthesizes calcitonin (thyroid) or calcitonin-gene related peptide (CGRP, brain). Even more complex is the alternative splicing that occurs in the α-tropomyosin transcript. At least 8 different alternatively spliced α-tropomyosin mRNAs have been identified. Abnormalities in the splicing process can lead to various disease states. Many defects in the β-globin genes are known to exist leading to b-thalassemias. Some of these defects are caused by mutations in the sequences of the gene required for intron recognition and, therefore, result in abnormal processing of the β-globin primary transcript. Patients suffering from a number of different connective tissue diseases exhibit humoral auto-antibodies that recognize cellular RNA-protein complexes. Patients suffering from systemic lupus erythematosis have auto-antibodies that recognize the U1 RNA of the spliceosome.