DNA Transcription and Translation PDF

Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub Transcription and translation Transcription takes place in three phases: initiation, elongation, and termination. First, the machinery that conducts transcription (a complex of proteins) needs to identify gene...

Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub Transcription and translation Transcription takes place in three phases: initiation, elongation, and termination. First, the machinery that conducts transcription (a complex of proteins) needs to identify genes and attach to DNA, a process called initiation. After this initiation, the transcription machinery will move along the DNA helix, elongating an RNA transcript as it goes. When the RNA transcript is complete, the machinery will disengage and release the RNA, terminating transcription. When you consider that the human genome is approximately 3 billion nucleotides long, and approximately 98.5% of that is noncoding, you wonder how cells are able to recognize where a gene begins. Specific DNA sequences, referred to as promoters, act to recruit proteins called transcription factors. Transcription factors are proteins that recruit and bind one of three RNA polymerases (I, II, III) to DNA. Transcription of protein coding genes is facilitated by RNA polymerase II, which binds to a Pol II promoter sequence in the genome. No single promoter element is absolutely required within the Pol II promoter to initiate transcription. Promoters consist of different combinations of a variety of elements, located upstream or downstream of the start site. Two examples of eukaryotic promoter elements are the CAAT and TATA boxes. As a means of orientation, the first transcribed nucleotide of a gene is referred to as the +1 site or start site (the start of transcription). The CAAT box is around 70-80 nucleotides upstream and the TATA box 25-30 nucleotides upstream of the gene’s start site. (The Pribnow box is an analogous promoter in prokaryotes.) Other important features of genes, eg, introns, exons, and untranslated regions (UTRs), will be discussed shortly. Promoter sequences are proximal regulatory sequences because they are located in close proximity to the gene they regulate. Although proximal promoter sequences are necessary for initiation of transcription, they are not sufficient. All Pol II dependent genes also require interaction with enhancer elements for efficient initiation and elongation. Distal regulatory sequences, which act as either an enhancer or silencer, can occur thousands of base pairs upstream or downstream from a gene. Enhancer sequences recruit activating transcription factors that promote the recruitment of RNA polymerase II, upregulating expression of the target gene and therefore the resultant protein. Silencer sequences operate in the same manner but instead recruit repressor factors that inhibit transcription, downregulating expression of the gene and the resultant protein. Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub When RNA polymerase II is properly engaged on the DNA helix, the elongation phase of transcription proceeds. RNA polymerases always travel in the same direction—from the 3’ end to the 5’ end of the template strand—pairing RNA nucleotides to the DNA strand and effectively elongating the RNA transcript one nucleotide at a time. (A key feature of RNA is that uracil, not thymine, is paired with adenine.) RNA polymerase II uses the template strand of the DNA helix to produce an RNA transcript that corresponds precisely with the other strand of the helix, referred to as the coding strand. While RNA polymerase moves in a 3’ → 5’ direction along the template strand, the RNA transcript, which is being elongated by adding nucleotides to its 3’ end, is actually being constructed in a 5’ → 3’ fashion. You may find the template strand referred to as the antisense strand, while the coding strand may be referred to as the sense strand. Once the RNA polymerase has fully transcribed the gene, transcription is terminated. The termination of transcription in eukaryotes is complex, and the exact mechanisms are still not fully described, but ultimately RNA polymerase II dissociates from the DNA helix and the RNA Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub transcript is released. Is the RNA transcript immediately ready to be translated into a protein? In eukaryotes, the answer is no. RNA Processing: the immediate product of transcription (hnRNA= heterogeneous nuclear RNA) requires medication before translation There are three important alterations to pre-mRNA that you need to be aware of: 5’ capping, the addition of a 3’-poly(A) tail, and intron splicing. RNA processing occurs co-transcriptionally, meaning the alterations just mentioned can be facilitated as the RNA transcript is still being produced. RNA processing begins with the addition of a 7-methylguanosine cap to the 5’ end of the pre- mRNA. This 5’ cap is added through an unusual 5’-to-5’ triphosphate bridge. The 5’ cap has several important functions: (1) nuclear pores recognize the 5’ cap and facilitate the export of mRNA into the cytoplasm, where translation occurs. (2) The 5’ cap protects mRNA from Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub degradation by 5’ exonucleases. (3) while in the cytoplasm, the 5’ cap also serves as an important initiating factor for translation. The 3’ end of pre-mRNA is modified by adding a poly(A) tail. At a distal portion of the pre-mRNA near the 3’ end, a polyadenylation sequence (5’-AAUAAA-3’) signals for a nearby downstream cleavage site. Following cleavage of the pre-mRNA at this point, addition of 40- 250 adenosine nucleotides to the pre-mRNA’s new 3’ end will take place. Poly(A) tail helps stabilize mRNA molecules. Poly(A)-binding proteins (PABP) associate with the poly(A) tail, shielding the RNA from exonucleases that facilitate degradation of the 3’ end of the pre-mRNA. The third modification of pre-mRNA that you need to be aware of is the removal of introns through a process called splicing. In general, exons are the sequences within a gene that are retained in mRNA and translated; introns are segments removed from pre-mRNA before translation occurs. Each intron has a 5’ donor site (5’-GU) and a 3’ acceptor site (AG-3’), as well as an internal adenine base referred to as the branch point. The protein complex that facilitates splicing, referred to as a spliceosome, is composed of several species of small nuclear ribonucleoprotein particles (snRNPs). The spliceosome first severs the donor site’s 5’ phosphodiester bond and establishes a new special 5’-2’ phosphodiester bond between the now free end of the intron and the internal branch point. This creates the characteristic lariat structure of removed introns. Next, the spliceosome facilitates the formation of a phosphodiester bond between the upstream exon’s free 3’ end and the downstream exon’s 5’ end. The intron, folded into a lariat, is released, and the spliceosome disassembles. Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub Alternative splicing: a process by which an organism is able to produce a range of unique but structurally related proteins (a transmembrane vs a secreted immunoglobin) from the same gene which can be alternatively spliced into a range of different mRNA molecules. Translation Once the mRNA is fully processed, it is exported from the nucleus into the cytoplasm, where it can undergo translation. Translation refers to the entire process by which mRNA is used as a template for the assembly of proteins. RNA sequences dictate the order and identity of amino acids following the genetic code, shown below (you should memorize AUG as codon start, and the 3 codon stop). Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub The cellular machinery involved in translation involves two additional types of RNA: ribosomal RNA (rRNA) and transfer RNA (tRNA). The ribosome is the primary functional unit of translation. It is a structure composed of two main subunits (each consisting of one or more rRNA molecules and multiple proteins). To fuel polypeptide production, tRNAs ferry amino acids into ribosomes for incorporation into the growing peptide chains. Ribosomes assemble on the 5’ end of mRNA with the assistance of helper proteins and initiation factors. A small 40S subunit combines with a larger 60S subunit to form an 80S ribosome. (In prokaryotes, 30S and 50S subunits create a 70S ribosome; these prokaryotic ribosomal units are Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub important pharmacologic targets for many antibiotics.) Each ribosome has three important sites. The A site is where charged tRNAs enter the ribosome, the P site holds onto the growing peptide chain, and the E site is where tRNAs are ejected from the ribosome, having had their amino acid already incorporated into the growing peptide chain. The only exception to this is the first amino acid of every protein, methionine, whose tRNA enters the ribosome at the P site. Transfer RNA (tRNA) molecules are used to carry amino acids into the ribosome in the correct order, according to the codon sequence of the mRNA. tRNA molecules have a 3’ stem dedicated to carrying a specific amino acid as well as an anticodon loop that will align with a specific codon in the mRNA. When a tRNA establishes a covalent bond with an amino acid, the tRNA is said to be charged. An aminoacyl-tRNA synthetase catalyzes the charging process using the energy of ATP. There is a unique aminoacyl-tRNA synthetase for each of the 20 amino acids to ensure accurate charging with cognate tRNAs. There are two other important functional sites in every tRNA, the T-arm and D-arm. Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub The T-arm helps the tRNA enter the ribosome, while the D-arm is used by the aminoacyl-tRNA synthetase to ensure that the correct amino acid is charged to each tRNA. Individual tRNAs are charged with a specific amino acid and only that amino acid. Because ribosomes lack proofreading capabilities, if tRNA’s are incorrectly charged (ie, if they are bound to an incorrect amino acid), the mischarged tRNA will enter a ribosome, pair with the codon correctly, but incorporate the incorrect amino acid into the elongating polypeptide. The incorporation of an improper amino acid has the potential to compromise the structure, and therefore the function, of the protein being produced. To initiate translation, a ribosome must form around a fully processed mRNA. A 40S subunit and a charged tRNA, with the assistance of initiation factors (IFs), slide down the mRNA’s 5’ end to locate the start codon (5’-AUG-3’). How is the 5’ end identified? The 40s subunit recognizes the 5’ cap, or an internal ribosomal entry site, to attach to the mRNA. Note, all nucleotides upstream of the start codon represent a segment of mRNA that is not translated, referred to as the 5’ untranslated region (5’ UTR). Because there is only one start codon within the genetic code, the first amino acid incorporated is always methionine; hence, met-tRNA is always used to initiate translation. Once the start codon has been identified, the 60S subunit joins to complete the ribosome. Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub With the ribosome in place, the elongation phase of translation can proceed. Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub The initial met-tRNA is positioned in the P site. According to the mRNA’s next codon, an appropriately charged tRNA enters the A site with the assistance of an elongation factor (a helper protein) by consuming the energy from one GTP molecule. The ribosome then catalyzes a peptide bond between the P site’s and A site’s amino acids. The new peptide bond anchors the peptide chain to the A site’s tRNA. The entire ribosome then shifts one codon down the mRNA in the 3’ direction, shifting the tRNA’s position within the ribosome. The newly vacated A site accepts the next charged tRNA as the uncharged tRNA now in the E site is ejected from the ribosome. As before, the ribosome catalyzes a peptide bond anchoring the peptide chain to the newest tRNA in the A site. The ribosome then shifts again, and the cycle continues as amino acids continue to be incorporated into the growing peptide chain. The elongation phase of translation continues in its cyclic fashion until the ribosome encounters a stop codon (5’-UAA-3’, 5’-UGA-3’, or 5’-UAG-3’). Instead of a charged tRNA, a release factor enters the ribosome’s A site and facilitates hydrolysis between the final amino acid and its tRNA Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub in the P site, leading to the disassembly of the ribosome and release of the newly synthesized peptide chain. Just like for tRNA, the entry of the release factor requires one GTP. Because the stop codon occurs before the 3’ end of the mRNA is encountered by the ribosome, all nucleotides downstream of the stop codon represent another untranslated region, the 3’ UTR. Proteins destination: Depending on where a protein is intended to ultimately function, translation occurs via ribosomes either freely floating around in the cytoplasm or via ribosomes associated with the rough endoplasmic reticulum (RER). Proteins that will ultimately function in the cytosol, nucleus, mitochondria, or peroxisome are translated by freely circulating ribosomes, as the protein will be released to its desired location. On the other hand, proteins that are destined to either be secreted, retained in the RER or Golgi, trafficked to the lysosome, or bound to the plasma membrane, are instead translated by ribosomes bound to the RER. Bound ribosomes conduct translation in the same manner as free ribosomes; in fact, they have the exact same structure. As a ribosome begins translation, the end of the peptide being synthesized is exposed to the cytoplasm. All peptides that require processing or transport through the RER Nucleic Acids: Structure and function Genetics Block Dr. Joelle Ayoub contain a signal sequence that is quickly recognized in the cytoplasm. A signal recognition particle (SRP) quickly makes the identification and will associate with the peptide bound to the ribosome (Figure 14). SRPs pause translation and help bind ribosomes to pores in the RER. Once the ribosome is bound, translation is resumed. Synthesis of the peptide is conducted through the RER pore, ensuring that on completion of translation, the peptide resides in the RER’s lumen. This bound ribosomal pathway helps ensure proteins that require packaging or transport (ie, digestive lysosomal enzymes) don’t end up circulating freely within the cytoplasm.