Module 4: From DNA to RNA

• gene = a genetic unit containing information to make a functional product (RNA and/or protein)
• genes contain:

  1. structural- coding DNA
  2. temporal- developmental
  3. positional- tissue/cell specific
  4. inducible- nutrients, stress, hormones
• Prokaryotic gene organisation:

  promoter- defines transcription start site and its direction

  leader/spacer- section of DNA which is nontranslated

  cistron- segment of DNA corresponding to one polypeptide (protein)
• Eukaryotic class II (mRNA-encoding) genes:

  enhancer- sometimes distal from promoter, contains transcription factor binding sites

  intron- in primary transcript but removed from mature transcript

  exon- in mature transcript

  UTR- untranslated region
• Eukaryotic genes are quite large ~ 50kB

  *most is non-coding

  *lower eukaryotic and prokaryotic genes are small snd equate more to the size of polypeptides they produce
• General mechanism of transcription:

  The DNA that contains a gene is made up of:

  1. coding strand (sense) 5'-3'
  2. has same sequence as RNA product
  3. template strand (antisense) 3'-5'
  4. sequence is complementary to RNA product
• Transcription "bubble"

  *RNA polymerase binds to DNA, melts double strand, polymerises 5' to 3' direction

  *RNA produced is copy of the coding strand and complementary to template strand
• transcription = the formation of a specific RNA sequence from a specific DNA sequence
• 1. RNA polymerase binds at the promoter and unwinds the double helix
  2. at the invitation site, RNA polymerase reads the DNA template strand and builds a complementary RNA strand of free nucleotide trio phosphates
  3. The RNA strand grows till its 3' prime end (elongation phase)

  -->RNA polymerase adds nucleotides in the 3' end

  4. The double helix rewinds as RNA polymerase moves through (requires energy)

  5. At the termination site, the RNA transcript is released from the template

  6.The DNA rewinds completely and the RNA polymerase dissociates from it (RNA polymerase is reusable)
• each type of RNA polymerase needs a distinct set of accessory factors
• Eukaryotes have 3 RNA polymerase:

  1. Pol I- ribosomal RNA genes (rRNA)
  2. Pol II- protein coding genes (mRNA), small nuclear RNA (snRNA)
  3. Pol III- transfer RNA (tRNA), rRNA and snRNA
• Assembly of RNA polymerase II initiation complex

  *TFIID = TATA binding protein (TBP) + TBP associated factors (TAFs)

  *TFIIA- helps TFIID to bind

  *TFIIB- sets distance from TATA element to start site

  1. interaction with D-A-B complex and recruitment of polymerase II
  2. blocks non-specific binding of polymerase II to DNA
• PROKARYOTES- transcription initiation

  1. RNA pol I needs a sigma factor to initiate transcription
  2. RNA pol binds to the promoter region (double stranded form- closed complex)
  3. DNA strands near transcription site starts to unwind to form an open complex
  4. RNA pol adds rNTPs onto template DNA (no primer)
  5. RNA pol produces short stretches of RNA which are released (occurs as the sigma factor blocks RNA exit channel of RNA pol)
• PROKARYOTES- transcription elongation

  1. sigma factor leaves the complex for the process of elongation
• PROKARYOTES- transcription termination

  RHO INDEPENDENT TERMINATION

  *no external proteins are required

  *intrinsic terminators consist of short inverted repeats (~20 nucleotides) and followed by AT rich sequence (8A:T)

  1. RNA pol transcribes the inverted repeat sequence (harpoon structure)
  2. Harpin structure halts RNA pol
  3. Transcription of the AT rich sequence results in AU base pairs (weakest base pairs)- RNA is released (ends transcription)
• PROKARYOTES- transcription termination

  RHO DEPENDENT TERMINATION

  *Rho protein required (hexametric protein- requires ATP)

  1. the rho protein binds to single stranded RNA rich in cytosine (known as Rut sites- Rho utilisation sites)
  2. when the RNA pol reaches 100 nucleotides away from the rut site- it stops the transcription (Rho sensitive pause site = sequence that halts transcription)
  3. once the rho protein binds to the rut sequence in RNA; it uses energy ATP to unwind the region
• EUKARYOTES- transcription initiation

  1. TFIID binds to TATA sequence in the promoter (40 nucleotides long and located upstream and downstream from the start site)
  2. TBP bends the DNA by 80- helps other transcription factors bind like TFIIA (stabilizies TFIID binding) and TFIIB (recruits RNA pol II)
  3. TFIIF is needed for RNA pol II to bind to the promoter
  4. TFIIE binds to the pre initiation complex
  5. TFIIF melts the promoter using ATP (creates open complex)
• EUKARYOTES- elongation

  *TFEb is attracted to RNA pol II by transcription activators

  -->kinase protein that phosphorylates serine residues in the C

  terminal domain of the polymerase- stimulates elongation

  *TFIIS helps increase the rate at slow regions (does not let RNA pol II to pause)

  *formation of mRNA occurs in the 5' prime to 3' direction
• EUKARYOTES- 5' Capping

  1. the terminal gamma phosphate of the nucleotide is removed by RNA triphosphatase
  2. Guanylyl transferase carries out the reaction between beta phosphate of the first nucleotide and the alpha phosphate of GDP
  3. Once guanine is attached, methyl transferase attaches a methyl group to the guanine nucleotide (called the 5' cap- helps mRNA bind to a ribosome for translation)
• EUKARYOTES- termination

  1. RNA polymerase reaches the end of the gene, the C-terminal domain of the RNA polymerase interacts with two proteins

  -->CstF= cleavage stimulation factor

  -->CPSF = cleavage and polyadenylation specificity factor

  2. once the end of the gene is transcribed into RNA, the proteins are

  recruited to mRNA by the C-terminal domain of RNA polymerase

  3. CstF leaves the mRNA

  4.Once the mRNA is cleaved, CSDF dissociates
• EUKARYOTES- termination [part 2]

  5.CPST 10 recruits Poly A polymerase which adds about 289 residues

  at the 3' end creating a poly A tail (requires ATP)

  6.A binding protein binds to the poly A tail, causing CDSF to be

  released from the mRNA

  -->the poly A binding protein prevents degradation of the poly A

  tail
• operon = cluster of genes transcribed by the same promoter that gives rise to polycistronic mRNA- genes usually related
• LAC OPERON

  *no lactose- lac operon is repressed

  -->repressor binds to O preventing RNA pol from cleating the

  promoter

  *negative feedback- active operon makes B-galactosidase, which hydrolyses lactose to glucose/ galactose, the operon switches off (self-regulating)
• cis-acting elements = will only regulate DNA to which it is directly joined to (dominant)
• trans-acting factor = will regulate genes anywhere (mostly protein transcription factors)(recessive)
• Role of CRP:

  *RNA pol requires CRP

  *cAMP binds to CRP permitting DNA binding

  *high glucose = low cAMP... so lac operon is off, even if lactose is available

  *low glucose = high cAMP...so lac operon is on if lactose is present

  *glucose preferentially used as a carbon source
• the trp operon:

  *contains genes for synthesis of amino acid tryptophan

  *trp repressor = binds to operator in presence of tryophan and prevents transcription

  *repressed by tryptophan (biosynthetic product)

  *induced by lactose (substrate)
• Regulation of transcription [part 1]

  *general transcription factors = proteins that bind to specific sites on DNA to activate transcription

  -->used with RNA pol and mediator multiple protein complex- which

  positions RNA pol at the start of a protein coding sequence

  *activators enhance the interaction between RNA pol and a particular promoter

  -->increase the attraction of RNA polymerase for the promoter via

  RNA pol subunits

  -->or by changing the structure of DNA
• Regulation of transcription [part 2]:

  *enhancers are sites on DNA that are bound to activators- to loop DNA in a specific way to bring a specific promoter (cis-acting)

  *repressors are proteins that bind to the operator, preventing RNA pol from transcribing

  *silencers are regions of DNA that are bound by repressor proteins to silence gene expression