RNA and Transcription PDF

RNA and Transcription Dr. Hilal Eren Gözel I. Okan University Nucleic Acids The two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), enable living organisms to reproduce their complex components from one generation to the next. DNA provides directions for...

RNA and Transcription Dr. Hilal Eren Gözel I. Okan University Nucleic Acids The two types of nucleic acids, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), enable living organisms to reproduce their complex components from one generation to the next. DNA provides directions for its own replication. DNA also directs RNA synthesis and, through RNA, controls protein synthesis. RNA The folded domains of RNA molecules may have catalytic capacities. Such catalytic RNAs are called ribozymes. Although ribozymes usually are associated with proteins that stabilize the ribozyme structure, it is the RNA that acts as a catalyst. Some ribozymes can catalyze splicing, a remarkable process in which an internal RNA sequence is cut and removed, and the two resulting chains then ligated. Types of RNA The vast majority of genes carried in a cell’s DNA specify the amino acid sequences of proteins. Some genes code for RNA molecules! Important RNAs for Transcription: Types of RNA *Telomeres are transcribed generating long non-coding RNAs known as TERRA! Transcription and Translation Genes provide the instructions for making specific proteins. A gene does not build a protein directly! The bridge between DNA and protein synthesis is the nucleic acid RNA. The flow of genetic information: DNA  RNA  protein Getting from DNA to protein requires two major stages: Transcription Translation  DNA 1 Synthesis of mRNA in the nucleus mRNA NUCLEUS CYTOPLASM mRNA 2 Movement of mRNA into cytoplasm Ribosome via nuclear pore 3 Synthesis of protein Amino Polypeptide acids Transcription and Translation Transcription is the synthesis of RNA using information in the DNA. The two nucleic acids are written in different forms of the same language, and the information is simply transcribed, or “rewritten,” from DNA to RNA. Translation is the synthesis of a polypeptide using the information in the mRNA via ribosomes in cytoplasm. Basic Principles of Transcription The simplest definition of a gene is a “unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA (such as a tRNA).” The vast majority of genes carry information to build protein molecules, and it is the RNA copies of such protein-coding genes that constitute the mRNA molecules of cells. During synthesis of RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed, into the four-base language of RNA, which is identical except that U replaces T. Basic Principles of Transcription During transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs, which then are joined in a polymerization reaction catalyzed by RNA polymerase. The mRNA nucleotide triplets are called codons and RNA molecules are always synthesized in the 5’3’ direction. Basic Principles of Transcription The transcription of a protein-coding eukaryotic gene results in pre-mRNA, and further processing yields the finished mRNA. The initial RNA transcript from any gene, including those specifying RNA that is not translated into protein, is more generally called a primary transcript. DNA polymerase vs RNA polymerase 1) RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides. 2) Unlike the DNA polymerases involved in DNA replication, RNA polymerases can start an RNA chain without a primer. This difference is thought possible because transcription need not be as accurate as DNA replication (more errors here!). 3) Unlike DNA polymerases, which make their products in segments that are later stitched together, RNA polymerases are absolutely processive; the same RNA polymerase that begins an RNA molecule must finish it without dissociating from the DNA template. Molecular Components of Transcription Steps of Transcription: Initiation of Transcription Elongation of the RNA Strand Termination of Transcription Initiation of Transcription The initiation of transcription is an especially critical process because it is the main point at which the cell selects which proteins or RNAs are to be produced. To begin transcription, RNA polymerase must be able to recognize the start of a gene and bind firmly to the DNA at this site. The way in which RNA polymerases recognize the transcription start site of a gene differs somewhat between bacteria and eukaryotes. Initiation of Transcription Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends. The DNA sequence where RNA polymerase attaches, and initiates transcription is known as the promoter; the sequence that signals the end of transcription is called the terminator. Initiation of Transcription The promoter, which contains a specific sequence of nucleotides that lies immediately upstream of the starting point for RNA synthesis. Once bound tightly to this sequence, the RNA polymerase opens up the double helix immediately in front of the promoter to expose the nucleotides on each strand of a short stretch of DNA. One of the two exposed DNA strands then acts as a template for complementary base-pairing. Transcription Chain elongation then continues until the enzyme encounters a second signal in the DNA, the terminator (or stop site), where the polymerase halts and releases both the DNA template and the newly made RNA transcript. This terminator sequence is contained within the gene and is transcribed into the 3ʹ end of the newly made RNA. Transcription Transcription The site at which RNA polymerase begins transcription is numbered +1. Downstream denotes the direction in which a template DNA strand is transcribed (or mRNA translated); thus a downstream sequence is toward the 3ʹ end relative to the start site, considering the DNA strand with the same polarity as the transcribed RNA. Upstream denotes the opposite direction. Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (-) sign. Initiation of Transcription in Prokaryotes In bacteria, it is a subunit of RNA polymerase, the sigma (σ) factor, that is primarily responsible for recognizing the promoter sequence on the DNA. Together, σ factor and core enzyme (RNA polymerase holoenzyme) complex adheres only weakly to bacterial DNA when the two collide, and slides rapidly along the long DNA molecule and then dissociates. However, when the polymerase holoenzyme slides into a promoter, the polymerase binds tightly, because its σ factor makes specific contacts with the edges of bases exposed on the outside of the DNA double helix (Step 1). Abortive Initiation 1. RNA polymerase binds to promoter DNA to form an RNA polymerase-promoter closed complex 2. RNA polymerase then unwinds one turn of DNA surrounding the transcription start site to yield an RNA polymerase-promoter open complex 3. RNA polymerase enters into abortive cycles of synthesis and releases short RNA products (contains less than 10 nucleotides) 4. RNA polymerase escapes the promoter and enters into the elongation step of transcription RNA polymerase- promoter closed complex  The tightly bound RNA polymerase holoenzyme at a promoter opens up the double helix to expose a short stretch of nucleotides on each strand (Step 2).  The region of unpaired DNA (about 10 nucleotides) is called the transcription bubble and it is stabilized by the binding of σ factor to the unpaired bases on one of the exposed strands.  The other exposed DNA strand then acts as a template for complementary base- pairing with incoming ribonucleotides, two of which are joined together by the polymerase to begin an RNA chain (Step 3). Transcription in Prokaryotes The first ten or so nucleotides of RNA are synthesized using a “scrunching” mechanism, in which RNA polymerase remains bound to the promoter and pulls the upstream DNA into its active site, thereby expanding the transcription bubble.  After abortive initiation, the core enzyme breaks free of its interactions with the promoter DNA (Step 4) and discards the σ factor (Step 5). Transcription in Prokaryotes At this point, the polymerase begins to move down the DNA, synthesizing RNA, in a stepwise fashion: the polymerase moves forward one base pair for every nucleotide added. During this process, the transcription bubble continually expands at the front of the polymerase and contracts at its rear.  Chain elongation continues until the enzyme encounters a second signal, the terminator (Step 6), where the polymerase halts and releases both the newly made RNA molecule and the DNA template (Step 7).  The free polymerase core enzyme then reassociates with a free σ factor to form a holoenzyme that can begin the process of transcription again (Step 8). Transcription in Eukaryotes In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three: RNA polymerase I, RNA polymerase II, and RNA polymerase III. The three polymerases are structurally similar to one another and share some common subunits, but they transcribe different categories of genes. RNA polymerases I, located in the nucleolus, transcribes genes encoding precursor rRNA (pre-rRNA), which is processed into 28S, 5.8S, and 18S rRNAs. RNA polymerase III transcribes genes encoding tRNAs, 5S rRNA, and an array of small, stable RNAs, including one involved in RNA splicing (U6) RNA polymerase II transcribes most genes, including all those that encode proteins. It also produces four of the five small nuclear RNAs that take part in RNA splicing. Transcription in Eukaryotes Although bacterial RNA polymerase and RNA pol II share similar structure, there are some differences: 1) While bacterial RNA polymerase requires only a single transcription - initiation factor (σ) to begin transcription, eukaryotic RNA polymerases require many such factors, collectively called the general transcription factors. 2) Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher- order forms of chromatin structure features that are absent from bacterial chromosomes. Transcription in Eukaryotes The general transcription factors help to position eukaryotic RNA polymerase correctly at the promoter, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter to start its elongation mode. The proteins are “general” because they are needed at nearly all promoters used by RNA polymerase II. They consist of a set of interacting proteins denoted arbitrarily as TFIIA, TFIIB, TFIIC, TFIID, and so on. *TFII standing for “transcription factor for polymerase II” Transcription in Eukaryotes The assembly process begins when TFIID binds to a short double-helical DNA sequence primarily composed of T and A nucleotides. For this reason, this sequence is known as the TATA sequence, or TATA box, and the subunit of TFIID that recognizes it is called TBP (for TATA-binding protein). The TATA box is typically located 25 nucleotides upstream from the transcription start site. It is not the only DNA sequence that signals the start of transcription, but for most polymerase II promoters it is the most important.  The binding of TFIID causes a large distortion in the DNA of the TATA box. This distortion is thought to serve as a physical landmark for the location of an active promoter in the midst of a very large genome, and it brings DNA sequences on both sides of the distortion closer together to allow for subsequent protein assembly steps.  Other factors then assemble, along with RNA polymerase II, to form a complete transcription initiation complex.  The most complicated of the general transcription factors is TFIIH. Consisting of nine subunits, it is nearly as large as RNA polymerase II itself and, performs several enzymatic steps needed for the initiation of transcription.  After forming a transcription initiation complex on the promoter DNA, RNA polymerase II must gain access to the template strand at the transcription start point.  TFIIH, which contains a DNA helicase as one of its subunits, makes this step possible by hydrolyzing ATP and unwinding the DNA, thereby exposing the template strand.  Next, RNA polymerase II, like the bacterial polymerase, remains at the promoter synthesizing short lengths of RNA until it undergoes a series of conformational changes that allow it to move away from the promoter and enter the elongation phase of transcription.  A key step in this transition is the addition of phosphate groups to the “tail” of the RNA polymerase (known as the CTD or C-terminal  domain) - TFIIH Additionally, the is a kinase! phosphorylation of the tail of RNA polymerase II causes components of the RNA splicing machinery to load onto the polymerase and thus be positioned to modify the newly transcribed RNA as it emerges from the polymerase. Transcription in Eukaryotes (Summary) After binding to a promoter, RNA polymerase separates the DNA strands in order to make the bases in the template strand available for base pairing with the bases of the ribonucleoside triphosphates that it will polymerize together. Cellular RNA polymerases melt approximately 14 base pairs of DNA around the transcription start site, which is located on the template strand within the promoter region. Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond. Elongation of Transcription After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter DNA and general transcription factors. During the stage of strand elongation, RNA polymerase moves along the template DNA one base at a time, opening the double-stranded DNA in front of its direction of movement and hybridizing the strands behind it. One ribonucleotide at a time is added to the 3’ end of the growing (nascent) RNA chain during strand elongation by the polymerase. Termination of Transcription During transcription termination, the final stage in RNA synthesis, the completed RNA molecule (primary transcript) is released from the RNA polymerase and the polymerase dissociates from the template DNA. Once it is released, an RNA polymerase is free to transcribe the same gene again or another gene. References & Thank You!