Chapter 13 - Transcription

RNA structure Nucleotides Ribose sugar – OH at 2′ C Nitrogenous bases Unstable; short-lived molecule Nitrogenous bases Adenine Guanine Cytosine Uracil

RNA structure Nucleotide polymer held together by phosphodiester bonds Usually single-stranded Due to short regions of complementary sequences, can base pair to form stems, hairpins, etc

RNA structure Primary structure Secondary structure Nucleotide sequence Secondary structure Formed by complementary regions Has greater variety than helix of DNA Various shapes have different functions

Classes of RNA Ribosomal RNA (rRNA) Messenger RNA (mRNA) Joins with protein subunits to form ribosomes Site of polypeptide synthesis Messenger RNA (mRNA) Codes for a polypeptide Amino acid sequence Pre-messenger/primary transcript In eukaryotic cells only Needs to be modified before exiting the nucleus Prokaryotic mRNA can start to be translated before transcription is complete Transfer RNA (tRNA) Brings specific amino acid to the ribosome for incorporation into the growing polypeptide

Classes of RNA cont Small nuclear RNA (snRNA) Joins with small nuclear proteins to form snRNPs – small nuclear ribonuclear proteins Assist with post-transcriptional modifications of primary transcript Splices out introns Small nucleolar RNA (snoRNA) Aids in the processing of rRNA

Classes of RNA cont MicroRNA (miRNA) and small interfering RNA (siRNA) In eukaryotic cells RNAi – RNA interference Initiates degradation or inhibition of mRNA molecules Piwi-interacting RNA (piRNA) Found in mammalian testes Regulation of sperm development

Synthesizing RNA from DNA During DNA replication, the entire DNA molecule is copied In transcription, only a small section of DNA is used for the synthesis of RNA Usually one gene at a time, or several genes (in prokaryotes) Only one of the two strands of DNA gets transcribed into RNA Transcribed/template strand Nontemplate strand = coding strand “coding” strand gives RNA sequence (replace T with U)

Synthesizing RNA from DNA cont In DNA, one strand may be the template strand for one gene, while another strand may be the template strand for another gene Transcription occurs in the 5′→3′ direction of the RNA molecule Complementary and antiparallel to the DNA strand

Transcription Unit Promotor RNA coding region Terminator Upstream from coding region Specific DNA sequence Serves as attachment site for transcription molecules Sequence is NOT transcribed into RNA RNA coding region Terminator Downstream from coding region Is transcribed into RNA; sequence is later removed Specific sequence to halt transcription

RNA polymerase Does NOT require a primer Prokaryotic RNA polymerase Single type of polymerase used for all transcription Composed of 5 polypeptide subunits – core enzyme (σ) sigma factor Binds with core enzyme to create holoenzyme Controls binding to promotor Without sigma, polymerase will bind anywhere on DNA Various sigma factors are present for different promotor types Releases from core protein after transcript is several nucleotides long Eukaryotic RNA polymerase Different classes for different types of RNA Consists of multiple subunits Core enzyme with accessory proteins at different stages

Bacterial transcription Initiation Specific DNA sequence at promotor Consensus sequence Most common nucleotides in a particular position R = purine Y = pyrimidine N = any

Promotor Two consensus sequences Any change/mutation in promotor region alters the rate of transcription Down mutation – reduces rate of transcription Up mutation – increases rate of transcription rare

Holoenzyme Binds to promotor consensus sequences only, but enzyme covers larger area Polymerase alters its structure and binds more tightly, unwinding DNA Begins at -10 sequence and continues downstream Bases on consensus sequence location, enzyme’s active site is in position +1 First RNA nucleotide is placed complementary to DNA sequence

Elongation After transcript is approximately 12 nucleotides long, polymerase structure alters so it is no longer bound to consensus sequences Moves downstream Sigma factor is usually released Polymerase continues to unwind DNA downstream and rewind upstream Transcription bubble Positive supercoiling ahead of bubble; negative supercoiling behind Topoisomerase enzymes relieve tension

Termination – Rho-independent Contains inverted complementary sequences that form a hairpin when transcribed Slows transciption 2nd repeat sequence is polyA (polyU on RNA) Weak (due to 2 H bonds between each), and transcript separates from DNA template

Termination – Rho-dependent Rho factor protein Binds to regions with no secondary structure RNA sequence upstream from termination doesn’t form secondary structure Rho factor binds to RNA and moves toward 3′ end At a hairpin, transcription slows and rho factor can “catch up” to DNA/RNA Rho has helicase activity Breaks H bonds and separates RNA from DNA

Modifications for eukaryotic transcription Nucleosome structure DNA associated with histone proteins Acetylation of histones reduces their positive charge; makes DNA more accessible Initiators Promotors Have varied sequences to attract different polymerase types Polymerases have several associated accessory proteins Directly upstream from gene Enhancers Can be located far away from gene DNA loops around to bring enhancer (with activator protein) to promotor region Some sequences can be repressors/silencers Termination Different polymerases have different mechanisms for termination