Partie 1 : Transcription

Introduction

• Reminders about the exam content

Exam 2

• The exam is on Thursday, March 20
• The content includes structure of the genome, chromatin and nucleosome
• The content includes mechanisms of transcription

Exam 3

• The exam is on Thursday, April 24
• The content includes RNA splicing
• The content includes regulation of transcription
• The content includes non-coding RNAs

Central Dogma

• Central dogma exception: retrotranscription (AIDS virus)
• ADN is copied into ADN m, then ADN m is translated into proteins
• Includes transcription and translation

Types of RNA

• Ribosomal RNAs (rRNA) constitute 80% of cellular RNAs
• Transfer RNAs (tRNA) make up 15% of cellular RNAs
• Messenger RNAs (mRNA) account for 3% of cellular RNAs
• Non-coding RNAs are never translated

RNA polymerase and Transcription: General Information

• RNA polymerases are key enzymes in transcription
• RNA polymerases are ADN-dependent
• RNA polymerase catalyzes gene transcription
• Uses one of the two ADN strands as a template
• Uses ribonucleotides (ATP, UTP, GTP, and CTP) as starting substrates
• Recognizes specific sites (promoter/terminator) that indicate the start and end of transcription
• The synthesis of RNA by RNA polymerase is quite accurate

Error rate

• The error rate is 1 error per 10,000 added nucleotides, important for speed
• ADN polymerase is 1000 times more accurate
• ADN polymerase has 1 error per 10 million added nucleotides
• ADN polymerase synthesizes ADN during replication

Genes to the to be transcribed

• Genes to be transcribed vary from cell to cell
• Genes to be transcribed vary based on the cell cycle
• Genes to be transcribed vary based on the environment
• Transcription is highly regulated to ensure the right amount, at the right place, at the right time
• Replication copies the entire genome, replication is not selective
• Transcription selectively copies certain parts of the genome at the right time

RNA strands

• Ribonucleotides are added to the template strand in the 5' to 3' direction
• An ARN-ADN hybrid is formed
• Bases are added according to their complementarity
• Chargaff’s rule applies
• Strands are antiparallel and complementary
• Ribonucleotide U replaces T

Coding Strand

• Identical to the ARN (except T vs. U)
• The newly synthesized ARN does not remain paired to the ADN template
• The ARN is released as the transcription bubble progresses

Matrix Strand

• Complementary and antiparallel

Promoters

• The template strand is not always the same

RNA Polymerases and Transcription: Structure, Homologies, and Distribution

• RNA polymerases are ubiquitous
• RNA polymerases are enormous protein complexes composed of multiple subunits
• The number of subunits varies by organism
• Exception: viral RNA polymerase (only one subunit)
• Five core subunits are conserved and directly involved in synthesis

Prokaryotes

• Have only one type of RNA polymerase

Eukaryotes

• Have three main RNA polymerases found in every eukaryote
• ARN pol I (rARN; except small 5s rARN)
• ARN pol II (mARN; most ncARNs except rARN and tARN)
• ARN pol III (tARN, 5s rARN, and certain ncARNs)
• Some groups have additional polymerases

Viruses

• Viruses also have RNA polymerases
• Viral RNA polymerases are simpler versions compared to those in other organisms

RNA Polymerase core

• The RNA Polymerase core of E. coli consists of 5 subunits
• Its quaternary structure has a molecular weight of approximately 400 kDa
• About 10 times larger than the average protein
• The lobe of the claw is stabilized by alpha 1 and 2, and by omega

Active Site

• The active site (shown in yellow) contains an essential Mg++ ion for catalysis
• Homologies in structural areas are particularly pronounced in internal portions
• Crab claws are formed by the two largest subunits

RNA Polymerases and Transcription Stages

• The three stages of transcription include: Initiation, Elongation, and Termination

Transcription Initiation Stage

• This step is critical for regulation and involves:
• Recognition of the promoter by the holoenzyme
• Complex closed formation
• Transcription bubble opening
• Complex open formation
• Initial transcription complex formation
• Aborted transcriptions
• Promoter evasion
• The initiation step is crucial and heavily regulated

Transcription Elongation Stage

• During elongation and synthesis, ARN polymerase:
• Unwinds double-stranded ADN ahead of the transcription bubble
• Winding single-stranded ADN behind
• Dissociates ARN transiently paired to the template strand
• Corrects transcription errors ('proofreading')
• About 14 bp are unwound to form the transcription bubble (open complex)
• Requires two Mg++ ions for the addition of nucleotides
• Only one magnesium is always associated with the polymerase to stabilize the intermediate phase of the nucleotide
• Speed is 50 to 90 nucleotides/s

Transcription Termination Stage

• Terminates with very specific sequences (terminator site]
• Includes synthesis arrest,
• Undocking of the ARN, ARN polymerase, and ADN,
• The final step is transcription bubble closure

Transcription in Bacteria

• The best studied model is Escherichia coli

RNA Polymerase Heart Role

• Role of Subunits
• Contains 2 alpha subunits:
  • They are necessary for assembly of the core
  • Necessary for recognition of the UP promoter CHEZ LES BACTERIES
• Contains 2 beta subunits:
  • Forms a crab claw to form the active site
  • Undergoes Stabilization and Assembly by omega subunit
  • Associates with sigma (σ) factor

Bacterial Transcription

• Bacterial Transcription occurs with an additional subunit Necessary for the Initiation sub-stage

Core Subunits

• The core synthesized ARN is not recognised by the Promotor

sigma Subunit

• Recongnition of the Promotor
• Is the Inititiation factor
• Undergoing transient association as part of the core, solely during the initiation step and phases, not the eongation phases

Holoenzyme Transcription

• Sigma Subunit requires Association with Cores subunits
• Transcribed as a non-functional transcription if not associated with the Apoenzyme
• Transcribed as Functional transcription because it recognises the Promotor

Transcriptional Variation

• In this step bacteria possesses different σ factors variation

Species Gene Recognition

• Specific recognitiion dependent on sigma factor variation of each species.
• Depending ont he Promotor sequences they favour transcription in different conditions

Genetic processes

• Genetic and Role regulation can occur via different sigma factiors.
• These processes are dependant on similar conditions with coordination

Bacterial Promotors

• Consists of:
  • Region of -10
• Bacterial Promotors most frequently contains : -35 regions
• And UP elements Upstream Promotor Element
• Non recognised by the factor σ which is soleley recognised by sub-unit σ

Bacterial Sites frequencey

• The -10 region, lacking -35 region
• Has a discriminating quality

Promotor and Sigma bacterial action

• Differing regions interact different Promotor elements.

Elements of Promoter

• Key elements of the promotor:
• Sigma 1, Sigma 2,2/3,4 Domain, σ1, σ2, σ2/σ3 and σ4
  • Each factor promotes promoter assembly

Bacterial Upstream sites

• Bacterial Promoters contain strong expressins for upstream presence
• This Upstream presence contains great concentration of A and T

Promoter sequence

• Spacing is important, presence of UP, important for recombination.
• Single Pair alteration can decreases efficancy.