Chapter 13 - The Genetic Code and Transcription

Central dogma

• The central dogma:

  • Transcription of DNA into RNA

  • Translation of RNA into protein

• Complications:

  • Not all DNA is transcribed into RNA (e.g., promoters, etc.)

  • Not all RNA is translated into protein (e.g., microRNA)

  • RNA is generally processed into its final usable form

  • Proteins are frequently modified into their mature forms

Learning Goals

• Be able to describe the functional roles for the different classes of RNA (rRNA, tRNA, mRNA)

• Understand the basics of transcription:

  • The mechanism for synthesis of RNA from a DNA template

  • The stages of transcription (initiation, elongation, termination)

  • Put transcription into the genomic context (where does it occur?)

• Understand the requirements for prokaryotic transcription:

  • DNA promoter elements (-10 and -35)

  • Proteins required: core RNAP and a sigma (σ) subunit

• Understand the requirements for eukaryotic transcription:

  • DNA promoter elements (TATA box)

  • The general transcription factors (GTFs) for RNA Pol II;

    • Focus on TFIID, one of the key factors (TFIID = TBP + TAF)

RNA polymerase directs the synthesis of RNA

• RNA is synthesized on a DNA template during transcription.

  • RNA molecules have multiple different functions

    • Coding: mRNA, tRNA

    • Catalytic, structural: rRNA

    • Regulatory: microRNA, snRNA

  • mRNA serves as the intermediate molecule between DNA and proteins.

    • It carries the information for the order of addition of amino acids during translation

RNA polymerase directs RNA synthesis

• RNA polymerase directs the synthesis of RNA using a DNA template. The catalytic reaction is similar to DNA synthesis by DNA polymerase.

  • Synthesis goes in the 5’ to 3’ direction, but no primer is required to initiate.

  • The enzyme uses ribonucleotides (NTPs).

  • The reaction for RNA synthesis can be expressed as:

Prokaryotic transcription

• Why start with prokaryotes?

  • Best understood → underlies all we know

  • Many features are conserved

  • Target of antibiotics

• There is one RNA polymerase (RNAP) that produces all types of RNA

  • The focus will be on mRNA synthesis

• Transcription requires three stages

  • Initiation, elongation (or synthesis), and termination.

    • No more steps; the mRNA is not modified after synthesis.

• Genes are usually arranged in polycistronic (multiple gene) operons

• Transcription and translation are coupled

  • Translation begins while the transcript is being made

Coupled transcription-translation

Transcription begins while the transcript is still being made

E. coli RNA polymerase

• There is one RNAP that is responsible for transcription of all RNAs (mRNA, tRNA, rRNA)

• RNA synthesis is in the 5’ to 3’ direction.

• RNA polymerase holoenzyme (whole enzyme) from E. coli contains core subunits and one that temporarily associates, the  (sigma) subunit.

  • The RNAP (RNA polymerase) core enzyme lacks sigma

    • the core enzyme consists of a2, b, b', w [don’t memorize]

  • The sigma subunit is responsible for promoter recognition during initiation. It is not present during elongation.

  • There are multiple sigma subunits that recognize different types of promoters under special circumstances

    • E.g.,, nitrogen starvation, stationary phase

Structure of E. coli RNA polymerase core enzyme

The crab claw

Sigma factors confer specificity

• Allows different genes to be expressed

• Some bacteria have many sigma factors, others only a few

Transcription of E. coli

Overview

• A) Transcription begins with promoter recognition and binding by RNA polymerase; closed complex.

• B) RNA synthesis begins at the transcription start site, where the DNA double helix is unwound to make the template strand accessible; open complex.

• C) Once initiation has been completed with the synthesis of the first 8–9 nucleotides, sigma () dissociates and elongation proceeds with the core enzyme.

Promoter recognition

First step: RNAP finds and recognizes promoters

• as dsDNA

• based on their DNA sequence

• through the σ subunit

Genome organization

• What does transcription look like in a genome?

• How does RNA polymerase know where to start the synthesis of an RNA molecule?

  • Recall: in replication, there was an origin of replication (a sequence of DNA) and proteins that were necessary to regulate where and when to start

  • Are there similarities? Is there a particular DNA sequence and special protein(s) that recognize it?

Prokaryotic (E. coli) genome organization

Operon - example

• Zoom in more….

• The RBS operon of E. coli (schematic view)

  • All 6 genes are transcribed on a single mRNA

  • Each of the genes has something to do with the metabolism (uptake, biosynthesis) of the sugar ribose

E. coli rbs operon (zoom in more) Sense strand shown; 1st half of operon

ORFs for first 3 genes shown in dark or light blue; start with ATG (the start codon) and end with one or two stop codons (TAG,TGA, TAA).

Very small intergenic space between ORFs (yellow): 5, 4, and 21 bases

Promoter (red): 5’-….TTGTCC …(15 bp)…TATACT….-3’; the single red A is +1 - where the transcript starts

Transcription initiation

• Initiation of transcription

  • Most E. coli promoters have two consensus sequences that comprise the promoter

    • The two sequences, TTGACA and TATAAT, are positioned at –35 and –10 with respect to the transcription initiation site.

    • What is a consensus sequence?

    • How was it discovered?

Identification of consensus promoter sequences

• Promoter regions of many genes are aligned by start site of transcription

• Found to have similar sequences at set distances

• Note: DNA sequences are the “top” strand

Identification of consensus promoter sequences

• Consensus sequences also show as logos (height indicates level of conservation)

• Confirmation - mutations reduce transcription

Transcription initiation

• Initiation of transcription

  • Most E. coli promoters have two consensus sequences that comprise the promoter

    • The two sequences, TTGACA and TATAAT, are positioned at –35 and –10 with respect to the transcription initiation site.

    • The consensus sequence is a sequence of DNA nucleotides most frequently found at each position and is necessary for a particular function.

  • These two parts together form the type of promoter that is recognized by the major sigma factor in the E. coli cell

    • The major sigma factor is named 70.

Operon - example (zoom out!)

• The RBS operon of E. coli

• All 6 genes are transcribed on a single mRNA: operon

  • Each of these genes has something to do with the metabolism (uptake, biosynthesis) of the sugar ribose

• The promoter is where RNA polymerase (RNAP) binds to start transcription (the answer to ‘ where to start?’)

  • transcription goes in one direction, synthesizing 5’ to 3’

Initiation and open complex formation

• The second step is for RNAP to bind to the promoter and partially unwind the DNA

• The template strand is in the active site

• The non-template strand is pushed out of the way)

Elongation (promoter escape)

• RNAP moves away from the promoter and into the coding region of the gene

• Releases σ

• The core enzyme synthesizes the RNA

Transcription elongation

• Synthesis of the growing RNA chain continues in the 5’ to 3’ direction.

  • The 3’ hydroxyl serves as the nucleophile to attack the a-phosphate of the incoming nucleotide (aka, nucleoside triphosphate).

  • The enzyme is highly processive but slower than DNA polymerase

    • RNAP goes at the rate of 40 nt/sec vs. 800 bp/sec for DNA polymerase III (ie., slower than DNA Pol III)

    • RNAP progresses at about the same rate as translation (the ribosome yields 15 aa/sec, moving at ~45 nt/sec).

Transcription - elongation

Transcription termination

• How does RNAP know when to stop?

  • Termination of transcription

    • There are two mechanisms to terminate transcription.

      • Rho-independent: At the end of the gene, transcription terminates due to hairpin formation in the RNA. This terminator, a rho-independent terminator, depends on the formation of a GC-rich stem-loop that is followed by a sequence of U

      • Rho-dependent: In some cases, termination depends on the rho (p) termination factor (i.e., a protein). Rho is a hexamer that uses ATP to dissociate the RNA from the DNA template.

Rho-independent terminators

• Rho-independent terminators

  • No protein is required; termination is based on the sequence of nucleotides in the RNA molecule.

  • The RNA folds into a stem-loop structure.

    • G-C rich stem

    • Run of U resides

  • Important point:

    • rU-rA bonds are stronger than rU-dA

    • Weakens the RNA interactions with DNA template → dissociation

Rho-dependent terminators

Rho factor: transcription termination in four steps

• Rho protein recognizes specific base sequences in the mRNA (called rut sequence).

• Rho is a hexamer that wraps RNA around itself.

• It uses the energy of ATP to pull the RNA away from the DNA template.

Think-pair-share question

• E. coli RNA polymerase, associated with the 70-kDal sigma subunit (sigma70), transcribes virtually all of the bacterial genome recognizing the consensus sequences of TTGACA at -35 and TATAAT at -10.

• Very shortly after infection of E. coli with the bacteriophage  (phi), transcription from the bacterial genome ceases and transcription from the phage is detected.

• Transcription of the phage genome requires the phage protein gp28 (a 28-kdal protein): in the absence of this protein no phage transcription is seen and normal E. coli transcription is evident. Although the gene that encodes gp28 has the consensus sequence described above, the rest of the phage genes whose transcription requires gp28 have an unusual promoter sequence:

  • AGGAGA at –35 and TTTNTTT at –10.

• Propose a mechanism for transcription that accounts for these data and include in your answer how transcription of gp28 occurs.

Extra slides: Transcription in E. coli

Extra slides: promotor recognition

Extra slides: a structural view of the initiation complex

Extra slides: open complex and initiation

Extra slides: elongation (promoter escape)