Transcription

Transcription Definition of a gene The entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide A cistron: a genetic unit that encodes a polypeptide(s) If it encodes one polypeptide, it is monocistronic If it encodes several polypeptides, it is polycistronic 173 Prokaryotic genes (operon) In bacteria, genes are polycistronic. In bacteria, genes that encode enzymes involved in related functions often are located next to each other The genes encoding the enzymes required to synthesize the tryptophan are located in one contiguous stretch This cluster of genes comprises a single transcription unit referred to as an operon. 174 Eukaryotic genes Most eukaryotic transcription units produce mRNAs that encode only one protein, thus termed monocistronic 175 Coding DNA in human genome Most of eukaryotic genomes contain non-protein coding regions In humans, for example, only 3% of the human genome contains protein-coding regions 176 Introns vs. exons The genomes of most eukaryotic cells contain specific DNA sequences that do not code for proteins These pieces of DNA are known as introns The coding regions are known as exons When RNA is synthesized, the RNA molecule contains both introns and exons and is known as precursormRNA (or pre-mRNA) 177 RNA splicing The intron sequences are removed from the newly synthesized RNA through the process of RNA splicing Now the RNA molecule is known as mRNA 178 Importance of introns The exon-intron arrangement may facilitate the emergence of new proteins How? 179 One… Introns allow genetic recombination 180 Alternative splicing The transcripts are spliced in different ways to produce different mRNAs and different proteins These are known as protein isoforms The process is known as alternative splicing 181 182 UDP-glucuronosyltransferase gene The 5' region of the UGT1A complex contains 13 tandemly arrayed first exons, (4 pseudo exons and 9 viable) Each first exon has its own promoter element Exons 2, 3, 4, and 5 are located in the UGT1A 3' region. All UGT isoforms contain the same C-terminal domain encoded by exons 2 through 5 The 9 first exons are independently spliced to exon 2 to generate 9 UGT1A transcripts The first exon determines substrate specificity The C-terminal region specifies interactions with UDPglucuronic acid 183 Splice variants for UDP glucuronosyltransferase 1 (UGT1A) Gene Where expressed Substrates UGT1A1 Biliary tissue, colon, intestine, liver, stomach Etoposide UTG1A3 Biliary tissue, colon, liver, stomach Genistein UGT1A4 Biliary tissue, colon, intestine, liver Tamoxifen UGT1A6 Biliary tissue, brain, colon, kidney, larynx, liver, lung, stomach PCBs UGT1A7 Esophagus, intestine, kidney, larynx heterocyclic amines UGT1A8 Colon, esophagus, intestine, kidney, larynx Benzo[a]phrene UGT1A9 Breast, colon, esophagus, liver, kidney, ovary, prostate, skin, testis Nicotine (UGT1A4) UGT1A10 Biliary tissue, colon, esophagus, intestine, orolaryngeal tissue, stomach Raloxifene 186 General description Transcription is the process of making RNA from DNA One of the two strands of the DNA double helix acts as a template for the synthesis of an RNA molecule 187 Using DNA strands Although both enzymes can read both DNA strands, RNA polymerase uses one strand at a time in order to make a RNA molecule. The transcribed DNA strand = template, antisense, (-) strand The other strand: sense, (+)strand, coding strand Gene 1 5’ 3’ Gene 2 Gene 3 3’ 5’ The strand used for transcription 5’ 3’ 5’ 3’ 3’ 5’ Gene 1 3’ 5’ Gene 2 Gene 3 3’ 5’ 5’ 3’ 188 Complementary sequences mRNA is complementary to DNA The RNA chain produced by transcription is also known as the transcript 189 Enzyme and substrate The enzymes that perform transcription are called RNA polymerases RNA polymerases catalyze the formation of the phosphodiester bonds between two nucleotides The growing RNA chain is extended in the 5-to-3 direction The substrates are nucleoside triphosphates (ATP, CTP, UTP, and GTP) 190 Energy A hydrolysis of highenergy bonds in NTP provides the energy needed to drive the reaction forward 191 Polysomes As RNA is synthesized, it is initially bonded to DNA, but after a short distance, the older polymerized RNA nucleotides are separated, and the newer ones become bonded This allows the simultaneous synthesis of many RNA chains from the same gene forming structures known as polysomes 192 DNA replication vs. transcription The RNA strand does not remain hydrogen-bonded to the DNA template strand RNA polymerase read the A in DNA and inserts U in the growing chain of RNA rather than T RNA molecules are much shorter than DNA molecules Unlike DNA, RNA does not store genetic information in cells 193 DNA polymerase vs. RNA polymerase RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides Unlike DNA polymerases, RNA polymerases can start an RNA chain without a primer RNA polymerases make about one mistake for every 104 nucleotides copied into the consequences of an error in RNA transcription are much less significant than that in DNA replication 194 Exonuclease activity Although RNA polymerases are not as accurate as the DNA polymerases, they have a modest proofreading mechanism 195 196 197 The RNA polymerase E. coli RNA polymerase is made up of multiple polypeptide chains The intact enzyme consists of four different types of subunits, called , , ', and  198 The core polymerase The core polymerase consists of two , one , and one ’ subunits The core polymerase is fully capable of catalyzing the polymerization of NTPs into RNA  is not required for the basic catalytic activity of the enzyme 199 Consensus sequences (the promoter) The DNA sequence to which RNA polymerase binds to initiate transcription of a gene is called the promoter A promoter is "upstream" of the transcription initiation site The region upstream of the transcription initiation site contains two sets of sequences that are similar in a variety of genes 200 The regions These common sequences are located approximately 10 and 35 base pairs upstream of the transcription start site They are called the (-10) and (-35) elements The transcription initiation site is defined as the +1 position Open reading frame: DNA sequence that can be transcribed into mRNA from first base to last one 201 How do we know they are important? Genes with promoters that differ from the consensus sequences are transcribed less efficiently than genes whose promoters match the consensus sequences Mutations introduced in either the -35 or -10 consensus sequences have strong effects on promoter function RNA polymerase generally binds to promoters over approximately a 60-base-pair region, extending from 40 to +20. The  subunit binds specifically to sequences in both the -35 and -10 promoter regions 202 Role of the  subunit In the absence of  , RNA polymerase binds to DNA with low affinity and nonspecifically The role of  is to identify the correct sites for transcription initiation and direct the polymerase to promoters by binding specifically to both the -35 and -10 sequences 203 Mechanism of transcription (initiation) The binding between the polymerase and a promoter is referred to as a closed-promoter complex The polymerase unwinds approximately 15 bases of DNA to form an open-promoter complex Single-stranded DNA is available as a template Transcription is initiated by the joining of two NTPs After addition of about the first 10 nucleotides,  is released from the polymerase 204 205 Mechanism of transcription (elongation) As the polymerase moves forward, it unwinds the template DNA ahead of it elongates the RNA rewinds the DNA behind it 206 Mechanism of transcription (termination) RNA synthesis continues until the polymerase encounters a termination signal where the RNA is released from the polymerase, and the enzyme dissociates from its DNA template 207 Termination sequences The simplest and most common type of termination signal in E. coli consists of a symmetrical inverted repeat of a GC-rich sequence followed by A residues Transcription of the GC-rich inverted repeat results in the formation of a stable stemloop structure 208 The effect of the stem loop structure The formation of this structure breaks RNA association with the DNA template, destabilizes the RNA polymerase binding to DNA, and terminates transcription 209 Rho-dependent terminator Rho is a helicase that follows the RNA polymerase along the transcript. When the polymerase stalls at a hairpin, Rho catches up and breaks the RNA-DNA base pairs, releasing the transcript. Rho-dependent termination signals do not have the string of U residues at the end of the RNA. 210 211 RNA polymerases In contrast to bacteria, which contain a single type of RNA polymerase, eukaryotic nuclei have three, called RNA polymerase I, RNA polymerase II, and RNA polymerase III RNA polymerase I transcribes rRNA genes RNA polymerase II transcribes protein-encoding genes RNA polymerase III transcribes tRNA genes and one rRNA gene 212 Eukaryotic RNA polymerases Eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes While bacterial RNA polymerase is able to initiate transcription without the help of additional proteins, eukaryotic RNA polymerases cannot. They require the help of a large set of proteins called general transcription factors 213 General transcription factors These general transcription factors help position the RNA polymerase correctly at the promoter aid in pulling apart the two strands of DNA to allow transcription to begin push the RNA polymerase forward to begin transcription 214 Why are they general? The proteins are "general" because they assemble on all promoters used by RNA polymerase II They are designated as TFII (for transcription factor for polymerase II), and listed as TFIIA, TFIIB, and so on 215 Mechanism of transcription (initiation) TFIID binds to a TATA box located upstream from the transcription start site The binding of TFIID causes a bend in the DNA of the TATA box This bend attracts other proteins to assemble on the promoter Along with RNA polymerase II, these protein factors form a transcription initiation complex One of them is TFIIH, which contains a DNA helicase. TFIIH creates an open promoter complex exposing the DNA template to the RNA polyemerase 216 217 Mechanism of transcription (elongation) Movement of the polymerase is activated by the addition of phosphate groups to the "tail" of the RNA polymerase. This phosphorylation is also catalyzed by TFIIH, which, also contains a protein kinase subunits 218 Mechanism of transcription (termination) Termination begins by stopping the RNA polymerase. There is a eukaryotic consensus sequence for termination, which is AAUAAA. After termination occurs, the transcript is released, and the phosphorylated Pol II is released from the DNA. The phosphates are removed by phosphatases, and Pol II is recycled for another round of transcription. Termination is coupled to the process that cleaves and polyadenylates the 3 end of a transcript. 219 Phosphorylation of RNA polymerase II RNA is processed and modified extensively Capping Splicing Polyadenylation Some of these processing proteins are associated with the tail of RNA polymerase II. These proteins jump from the polymerase tail onto the RNA molecule as it appear. 220 Addition of a cap As soon as RNA polymerase II has produced about 25 nucleotides of RNA, the 5' end of the new RNA molecule is modified by addition of a "cap" that consists of a modified guanine nucleotide The guanine is added in a reverse linkage (5’ to 5’ instead of 5’ to 3’) 221 Importance of capping The 5’-methyl cap signals the 5’ end of eukaryotic mRNAs this helps the cell to distinguish mRNAs from the other types of RNA molecules, which are uncapped In the nucleus, the cap binds a protein complex called CBC (cap-binding complex), which helps the RNA to be exported into the cytoplasm The 5’-methyl cap also has an important role in the translation of mRNAs to proteins 222 RNA splicing The machinery that catalyzes pre-mRNA splicing consists of 5 RNA molecules and over 50 proteins. The RNA molecules are known as snRNAs (small nuclear RNAs) Each one of them is complexed with protein subunits to form a snRNP (small nuclear ribonucleoprotein) These snRNPs form the core of the spliceosome, the assembly of RNA and proteins that perform pre-mRNA splicing The catalytic site itself is largely formed by RNA molecules instead of proteins 223 hnRNP Another class of proteins that assemble on pre-mRNA is hnRNPS (heterogeneous nuclear ribonuclear proteins) hnRNP particles bind to introns They have different functions 224 Accuracy of splicing The consistent exon size (more uniform than introns) The assembly of the spliceosome occurs as the premRNA emerges from the RNA polymerase II As RNA synthesis proceeds, spliceosome components, called the SR proteins, mark the 3’ and 5’ splice site hnRNPs define introns Spliceosome assembly is co-transcriptional, but splicing occurs post-transcriptionally 225 Polyadenylation A certain sequence in the mRNA (AATAA) in the 3’ ends of mRNAs is recognized by RNA-binding proteins and RNAprocessing enzymes that cleave the RNA. Poly-A polymerase adds ~200 A nucleotides to the 3’ end produced by the cleavage. The nucleotide precursor for these additions is ATP 226 Poly-A polymerase Poly-A polymerase does not require a template hence the poly-A tail of eukaryotic mRNAs is not directly encoded in the genome 227 Poly-A-binding proteins Poly-A-binding proteins bind to the poly-A tail Help in transporting mRNA from the nucleus to the cytosol Help in protein synthesis Stabilize mRNA 228 Alternative polyadenylation • Many protein-coding genes have more than one polyadenylation site, producing mRNAs with different lengths of a noncoding sequence at the 3’-end called the 3‘-untranslated region (3’-UTR). 229 Alternative polyadenylation • The choice of poly(A) site can be influenced by extracellular stimuli that regulate the expression of the proteins that take part in polyadenylation. • Having a shorter transcripts would remove regulatory elements in the 3‘-UTR and influence the half-lives of mRNA and, hence the amount of generated proteins. • Example: Longer 3’UTR would contain binding sites for microRNAs at the 3‘-UTR, which tend to repress translation and promote degradation of the mRNAs. 230 SNPs and alternative polyadenylation The presence of SNPs within the polyadenyation signal can also alter the length of the mRNA and , hence, protein amount in cells. 231 SNPs in promoter • Single nucleotide polymorphisms (SNP) in promoter region can alter the binding of transcription factors required for the expression of a gene. • These variations may increase or decrease the expression of the affected gene, which eventually can influence the risk of developing a disease. mRNA transport Transport of mRNA from the nucleus to the cytoplasm, where it is translated into protein, is highly selectiveand is associated to correct RNA processing Defective mRNA molecules like interrupted RNA, mRNA with inaccurate splicing, and so on, are not transported outside the nucleus 233 Degradation of mRNAs The vast majority of mRNAs in a bacterial cell are very unstable, having a half-life of about 3 minutes The mRNAs in eukaryotic cells are more stable (up to 10 hours; average of 30 minutes) Exonucleases are responsible for degradation 234 235 Iron-responsive elements • In human cells, there are regions of mRNA called iron responsive elements (IREs) • These regions are contained within the mRNA sequences that code for certain proteins that regulate the levels of iron • Ferritin, transferrin receptor, ferroportin, and DMT1 • Iron responsive element binding protein (IRE-BP) binds to these mRNA sequences influencing protein expression 236 Effect on expression When iron is abundant, it binds to IRE-BP, disabling the binding of IR-BP to ferritin mRNA This prevents the degradation of the mRNA molecules allowing the production of more ferritin protein Therefore, the iron itself causes the cell to produce more iron storage molecules On the other hand, at low iron levels, the IRE-BP will bind to the ferritin mRNA and, thus, the mRNA will be destabilized, making less ferritin protein An opposite effect is seen on the stability of transferrin receptor mRNA 237 238 239 Gene amplification It is an increase in copy number of a restricted region of a chromosome increasing the is the quantity of DNA in these regions. It is a mechanism that cancer cells use to escape resistance from methotrexate whereby the target gene, dihydrofolate reductase, is amplified. It is also a mechanism by which breast tumor cells progress and become more aggressive whereby they amplify the human epidermal growth factor receptor 2 (HER2), which stimulates cell growth.