Transcription

• What is DNA transcription?

  RNA synthesis
• What is a transcript?

  product of tanscription (mrna)
• What are the similarities of transcription and replication?

  Template dna: doesnt participate in the reaction but defines which substrate is gonna be incorporated

  
  

  In each reaction a NTP is added to a polynucleotidic chain

  
  

  PPI hydrolysis provides the Gibbs energy needed

  
  

  RNA synthesis is catalyzed by RNA polymerase with a similar mechanism to that of DNA polymerase in 5-3 direction
• What are the differences between transcription and replication?

  RNA polymerase: no need of primers , no 3-5 exonuclease activity , higher mistakes rate , slower

  
  

  only one strand of DNA is transcribed each time

  
  

  only some sequences of DNA are transcribed (constitutive , inducible)

  
  

  Each gene is transcribed many times and not once
• How to name DNA strands?

  In each gene the nontemplate strand: coding strand (same sequence than RNA) or + strand

  
  

  Template strand is the noncoding strand and symbolized as -
• How to locate genes?

  In each gene the first nucleotide to be transcribed is given number +1 , DNA sequences upstream to the starting point have negative values and downstream positive values

  Sequences are given according to those in the nontemplate strand
• What are the characteristics of promoters?

  DNA sequences or elements upstream of the transcription starting point , they have specific positions and consensus sequences , core promoter TATAAT box (-10) and TTGACA box (-35)
• What are the characteristics of RNA polymerase?

  no need of primers , no 3-5 exonuclease activity , higher mistakes rate , slower , one RNA polymerase synthesizes all RNAs

  Holoenzymes: core a2 , b ,b' , w (for RNA synthesis) ans sigma subunit (promoter recognition)
• How is the promoter recognized?

  Sigma identifies and binds the core promoter sequences
• What is the first step of transcription?

  Initiation:

  RNA polymerase subunit with helicase activity(opens double-stranded bonds) (transcription bubble) recognizes sequences dsDNA , different types of sigma recognize different promoter.

  
  

  RNA polymerase moves along the strands and stops when sigma recognizes box. RNA polymerase synthesises RNA , if RNA polymerase introduces 8 bases then sigma factor detaches and RNA polymerase continues to move down
• What is the second step of transcription?


  Elongation:

  Nucleotides are added at a rate of 50-90nc/sec , topoisomerases relive the strain caused by transcription (Unwind negative supercoils) , elongation proceeds with slow downs and detentions
• What is the last step of transcription?

  Termination:

  both transcription initiation need to be controlled , termination signals are in RNA , two ways of termination:

  Rho factor independent termination

  Rho factor dependent termination
• What occurs during Rho independent termination?

  Termination signal is a stretch of 30-40 nucleotides on the RNA transcript consisting of many GC followed by a series of UA . A fork is formed in nascent RNA due to autocomplementary sequences , fork is destabilizes the double helix and the RNA polymerase leaves DNA
• What occurs during Rho dependent termination?

  Rho is a hexamer of identical subunits with helicase activity , Rho factor binds to the Rut site in single stranded RNA , it moves along the newly formed RNA and separates the mRNA from the dna template
• How is transcription regulated in Prokaryotes?

  proteins with related functions are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter resulting in the formation of polycistronic transcript. Nutrients or other molecules have a direct effect in regulation , few promoter elements and few proteins involved
• What are the types of regulation?

  negative regulation:

  Lactose operon , tryptophan operon

  
  

  Positive regulation: arabinose operon , activator is bound to promoter to express gene
• What are the characteristics of the lactose operon?

  For the expression of genes to acquire and metabolize lactose ,

  
  

  in the absence of lactose , the lac repressor binds the operator and transcription is blocked. In the presence of lactose , the lac repressor is released from the operator and transcription proceeds at a slow rate
• What are the characteristics of the tryptophan operon?

  For the expression of genes to synthesize tryptophan, in the absence of tryptophan , the trp repressor dissociates from the operator and RNA synthesis proceeds , When tryptophan is present the trp repressor binds the operator and rna synthesis is blocked
• How is RNA processed?

  In prokaryotes RNAs are polycistronic , mRNAs are short lived and translated as soon as synthesized
• How is DNA transcribed in eukaryotes?

  Three different RNA polymerases : 1 (rRNA) , 2 (mrna precursors and snRNA) and 3 (tRNA and rRNA)

  
  

  GTFs (general transcription factors): each polymerase requires them to recognize promoters and be fully effective
• What are transcription factors?

  each polymerase require them to recognize promoters and be fully effective , TATA (-30)

  TFIIH (last to bind)
• How is transcription regulated in eukaryotes?

  promoters are more complex , many regulatory transcription factors and cofactors control gene expression , chromatin modifications are involved in transcriptional regulation , positive regulation mechanisms are more frequent than negative ones
• How is RNA processed in eukaryotes?

  5' cap (to protect 5' end we add 7 methylguanosine) , 3- poly A tail (not coded in DNA) , splicing (non coding removed)
• What are the characteristics of splicing?

  Takes place in the nucleus , some introns can be removed by an autocatalystic process (nucleotides in mRNA carry out the reaction) , most of introns require of the catalytic activity of nucleoproteins organized in a spliceosome