Transcription How the Information in DNA Is Used to Produce RNA in Prokaryotes and Eukaryotes

Information Flow From DNA replication DNA transcription RNA translation Protein

Components of RNA Nucleotides Purine bases Adenine (A) Guanine (G) Pyrimidine bases Cytosine (C) Uracil (U) 5-carbon sugar Ribose Phosphate PO4

RNA Structure Primary Structure Chain of Nucleotides Secondary Structure Single folded chain RNA to RNA base pairing rules: G pairs with C A pairs with U

Base pairing creates the unique secondary structure of transfer RNA molecules.

Transcription Transcription = production of RNA using DNA as a template One DNA strand is used to produce a RNA strand, beginning at a promoter and ending at a terminator. Base in DNA template Base in RNA product A U T G C

Structure of Transcribed Region

Transcription 3’---TACAAA GAGACT---5’ DNA template Transcription requires 1. RNA polymerase, an enzyme that adds nucleotides in a 5’3’ direction. 2. Nucleoside triphosphates 3. Energy: release of diphosphate 3’---TACAAA GAGACT---5’ DNA template 5’---ATGTTTCTCTGA---3’ Antisense strand 3’---TACAAAGAGACT---5’ 5’---ATGTTTCTCTGA ---3’ 5’---AUGUUUCUCUGA---3’ mRNA Sense strand

Different Genes Can Have Different Template Strands For each gene, only one of the two DNA strands is used as a template.

Applying Your Knowledge What is the sequence of an RNA molecule transcribed from a DNA template strand that reads 3’-ATG-5’? 5’-TAC-3’ 5’-CAU-3’ 5’-AUG-3’ 5’-UAC-3’ 3’-TAC-5’

Stages of Transcription Initiation transcription apparatus binds to promoter Elongation RNA polymerase adds nucleotides to chain Termination recognition of termination signal separation of RNA from DNA template

Features of the Prokaryotic Promoter

Two Forms of RNA Polymerase in Prokaryotes Holoenzyme = Core enzyme + Sigma factor Recognizes –10 and –35 regions Binds to promoter Forms open promoter complex Core enzyme continues transcription after initiation

Initiation Holoenzyme forms Holoenzyme binds to promoter Open promoter complex forms First RNA nucleotide binds

Elongation RNA nucleotides are added in a 5’3’ direction by RNA polymerase core enzyme.

Rho-Dependent Termination Rho-Dependent terminators have region without secondary structure sequence that produces a pause in transcription Rho protein binds to region without secondary structure Terminator sequences form hairpin to slow polymerase Rho travels to terminator region and unwinds RNA-DNA duplex

Rho-Independent Termination Rho-independent terminators have inverted repeats string of adenines When terminator is transcribed inverted repeats form hairpin that slows RNA polymerase A-U pairs are unstable RNA separates from template

Eukaryotic Transcription Different types of RNA are produced by different polymerases. Eukaryotic RNA Polymerase Messenger RNA (mRNA) RNA Polymerase II Ribosomal RNA (rRNA) RNA Polymerase I Transfer RNA (tRNA) RNA Polymerase III

Features of the Eukaryotic RNA Polymerase II Promoter General Transcription Factors + RNA Polymerase II bind to Core Promoter Transcriptional Activator Proteins bind to the Regulatory Promoter

Initiation of Eukaryotic Polymerase II Transcription Transcription Factor IID binds to TATA box on core promoter Holoenzyme forms and binds to TFIID Holoenzyme = RNA Pol II + Transcription Factors Basal Transcription Apparatus

Initiation of Eukaryotic Polymerase II Transcription Basal Transcription Apparatus is needed to support minimal levels of transcription. Transcription can be increased by Transcriptional Activator Proteins bound to Regulatory Promoter Enhancer Sequence

Termination of Eukaryotic Polymerase II Transcription exonuclease Termination of Eukaryotic Polymerase II Transcription Polymerase II continues transcription beyond the coding region Cleavage occurs at the 3’ end, near consensus sequence Rat1 exonuclease binds to 5’ end and moves toward Polymerase II, degrading the RNA from 5’3’ Transcription terminates when Rat1 reaches Polymerase II

Transcription Termination for Eukaryotic RNA Polymerases I and III RNA Polymerase I requires a termination factor that binds to a DNA sequence downstream of the termination site RNA Polymerase III transcribes a terminator sequence to produce a region rich in uracil residues on the transcript Pol I is similar to rho-dependent termination except that the terminator binds to DNA rather than RNA. Pol III is similar to rho-independent termination except that a hairpin does not precede the string of uracils.

Applying Your Knowledge Prokaryotes Eukaryotes Both Prokaryotes and Eukaryotes Neither Prokaryotes nor Eukaryotes Which organism(s) have A. Sequences at –10 and –35 that are recognized by Holoenzyme? B. Transcriptional Activator Proteins? C. Rho-Dependent Termination? D. Rat1 exonuclease activity?