Lecture 4 (1) PDF - Transcription & RNA Processing

Transcription and RNA Processing Pierce Chapters 13, 14 Computer model of eukaryotic RNA polymerase II  Section Outline From Chapter 13… 1.Basic features of RNA 2.The Process of Gene Expression 3.Tra...

Transcription and RNA Processing Pierce Chapters 13, 14 Computer model of eukaryotic RNA polymerase II  Section Outline From Chapter 13… 1.Basic features of RNA 2.The Process of Gene Expression 3.Transcription in Prokaryotes 4.Transcription in Eukaryotes From Chapter 14… 5.RNA molecules and RNA processing  Components for DNA replication I A B C D D E E F G B H DNA ligase DNA gyrase DNA polymerase I Leading strand DNA polymerase III Lagging strand Primase RNA Primer DNA Helicase DNA polymerase I binding site Replication Origin DNA polymerase III binding site  Components for DNA replication DNA ligase DNA polymerase I DNA polymerase III 1. The Central Dogma of Molecular Biology  1. Basic features of RNA Recall… -RNA contains the base uracil in place of thymine -RNAhas a ribose sugar (it bears a hydroxyl (–OH) group on its 2’ carbon) RNA has tertiary structure (example: tRNAs). RNAs may interact as functional units (quaternary structure) (example: in the ribosome). DNA can also display these structural features but much more limited. Therefore, in some ways, the various structures of RNA can resemble proteins!  1A. Basic features of RNA Why Is RNA Less Stable Than DNA And Why Use It? The presence of the unique 2’OH group in ribose causes it to react intramolecularly with the 3’OH site resulting in phosphate bond breakage  1A. Basic features of RNA Why use an unstable RNA? Partly is a carry over from evolution – RNA evolved first Partly because RNA can form many tertiary structures allowing it to have different conformations for different functions, whereas DNA is generally only double stranded Partly because a temporary and degraded molecule offers a way of controlling its level (for example shutting off expression) 1A. Basic features of RNA  1A. Basic features of RNA See also Table 13.2 https://www.youtube.com/watch?app=desktop&v=FThA4Vxs3v4  2. The Process of Gene Expression Transcription: synthesis of RNA from DNA templates All Cellular RNAs are synthesized from DNA templates  1. Basic features of RNA We will focus on transcription of mRNAs See also Table 13.2 https://www.youtube.com/watch?app=desktop&v=FThA4Vxs3v4  2. The Process of Gene Expression Transcription is exhibited as this Christmas tree like structure  2. The Process of Gene Expression RNA is synthesized in the 5’ to 3’ direction using the 3’to 5’ DNA template strand. RNA synthesis is complementary and anti-parallel to the DNA template strand.  2. The Process of Gene Expression There are multiple genes on chromosomes and they can be located on either DNA strand as shown for genes a, b, c below. Therefore, transcription can utilize either DNA strand as the 3’ to 5’ template, but transcription always occurs in the 5’ to 3’ direction  2. The Process of Gene Expression Components needed for Transcription 1. DNAtemplate 5’ 3’ 2. 4 ribonucleosidetriphosphates (rNTPs) A, U, C, G RNAn + rNTP RNAn+1 +PPi 3. DNAdependant RNA polymerase NOTE: the use of ribonucleoside 3’ 5’ triphosphates in transcription rather than deoxynucleoside triphosphates  2. The Process of Gene Expression General features of RNA synthesis Similar to DNA Synthesis except – The precursors are ribonucleoside triphosphates (rNTPs) – Only one strand of DNA is used as the template (the 3’ to 5’ strand). – As we will see, RNA chains can be initiated de novo (no primer required). The 5’ to 3’ RNA molecule will be complementary to the DNA template 3’ to 5’ strand The 5’ to 3’ RNA molecule will be similar (except that uridine replaces thymidine) to the DNA non-template 5’ to 3’ strand (also knows as the DNA sense). RNA synthesis is catalyzed by RNA polymerases and ALWAYS proceeds in the 5’➔3’ direction.  2. The Process of Gene Expression Generalized structure of a prokaryotic gene 3. Transcription in Prokaryotes RNA Polymerase Holoenzyme Only 1 RNA polymerase in prokaryotes Functions of the subunits: : (alpha) -involved in the assembly of the tetrameric core (2 of these) : (beta) – contains the ribonucleoside triphosphate (rNTP) binding site ’: (beta-prime)- contains the DNA template binding region ω : (omega)-it helps to stabilize the tetrameric (2  ’) core : (sigma) – it binds to the RNA polymerase tetrameric core and assists in the correct initiation of transcription specifically at the promoter region of the prokaryotic gene. Many types of sigma factors → allows for specificity  3. Transcription in Prokaryotes Sigma factor () recognizes and binds to the -35 and -10 consensus sequences in the promoter region, properly positioning the RNA polymerase to begin transcription. The -10 consensus sequence is prone to unwinding due to its AT rich content.  3. Transcription in Prokaryotes Initiation Once the holoenzyme has bound to the promoter, RNA polymerase is positioned over the transcription start site (at position +1) and has unwound the DNA to produce a single-stranded template. The orientation and spacing of the consensus sequences on a DNA strand determine which strand will be the template for transcription and thereby determine the direction of transcription.  3. Transcription in Prokaryotes Initiation RNA polymerase binds, unwinds and joins first 2 nucleotides. Initiation of RNA synthesis DOES NOT require a primer  3. Transcription in Prokaryotes Elongation Elongation occurs when Sigma factor is released and RNA polymerase begins to move along the 3’ to 5’ DNA template strand.  3. Transcription in Prokaryotes Elongation Complementary nucleotides continue to be added during the elongation process. Localized DNA unwinding ahead of RNA polymerase generates a “transcription bubble”. Transcription bubble moves with the RNA polymerase and the unwound DNA rewinds behind it. RNA polymerase has both helix unwinding and rewinding activities  3. Transcription in Prokaryotes Termination Transcription stops when RNA polymerase reaches the “terminator” region of the gene. Occurs upstream of where the actual termination will take place. The newly-synthesized RNA together with the RNA polymerase are released. Bacterial cells possess two major types of terminators: Rho-dependent (requires Rho factor) and Rho- independent (aka intrinsic terminator) 3. Transcription in Prokaryotes Rho-dependent Termination Two sequence features: 1) DNA sequence of terminator site causes Polymerase to pause 2) DNA sequence upstream of terminator encodes a stretch of RNA that is C rich and devoid of secondary structure. a) Called the rho utilization (rut) site. b) Rho binds to rut site  3. Transcription in Prokaryotes Rho-dependent Termination Rho moves along RNA towards paused Polymerase Rho factor has helicase activity Unwinds the RNA-DNA hybrid Brings transcription to an end 3. Transcription in Prokaryotes Rho-independent Termination Makes up 50% of all terminations in prokaryotes. 2 common features: 1) contains inverted repeats 2) A string of 6-9 A’s follows the inverted repeats 3. Transcription in Prokaryotes Rho-independent Termination Poly A sequence is transcribed into a poly U tail after the hairpin is transcribed. Causes polymerase to pause Hairpin forms and destabilizes the DNA- RNA hybrid Assisted by the weak A- U base pairing Which statement is NOT correct about the process of transcription? a. Transcription of a given gene typically takes place on only one of the two DNA strands. b. During transcription, the RNA molecule is synthesized in the 3´-to-5´ direction. c. During transcription, the RNA molecule that is synthesized is antiparallel and complementary to the template DNA strand. d. The start site and direction of transcription are determined by a region known as the promoter. e. The RNA molecule that is synthesized will have U in place of T.