Transcription (RNA synthesis)

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Central Dogma of Molecular Biology
It describes gene expression from DNA to proteins; the flow of information in the cell:
DNA is transcribed into RNA which is translated to Proteins.
Some Basics of Transcription
It is the 1st step in gene expression (making the product of a gene). Proteins perform most cellular functions. Transcription is similar to DNA replication except that RNA is produced instead of DNA, only selected genes are copied (and only 1 strand of a gene is transcrubed, template strand), uses RNA polymerase (instead of DNA polymerase) and only one strand of DNA is used for any given gene but strand varies for different genes.
DNA is localised in the nucleus and proteins are synthesized in the cytoplasm so RNA basically transfers information. How we know? Pulse chase with radioactive uracil, RNA is synthesized in nucleus but moves into the cytoplasm. Pulse chase shows expts show that RNA synthesis is transient when a phage virus infects E.coli bacteria (volkin and astrachan 1957)
RNA (ribonucleic acid) is found mainly in the cytoplasm, carries out the genetic instruction for protein synthesis.
There are 4 types distinguished by relative size, shape and specific role => in carrying out DNA instruction in protein synthesis:
- mRNA (messenger)
- tRNA (transfer)
- rRNA (ribosomal)
- snRNA (small nuclear)
(- viral RNA)

DNA structure has 2 anti-parallel strands (double stranded)
Upstream: 5' on coding strand and 3' on template strand and Downstream is 3' on coding and 5' on template strand.    
(In transcription) Genes are read in a 3' to 5' direction from template strand. Nucleotide is added to growing single stranded chain of mRNA only at 3' end. Template strand is read in a 3' to 5' direction and the new RNA strand is synthesized in a 5' to 3' direction and the new RNA strand has identixal sequence (with U replacing T) to the untrascribed strand - the coding strand. And as mentioned, different genes can be transcribed from different strands of dsDNA but transcription is ALWAYS in 5' to 3' direction.
RNA polymerase unwinds the DNA helix to form a bubble and reads the template strand (as mentioned) in a 3' to 5' direction. The RNA is formed in a 5' to 3' direction and displaced from template as the bubble closes. The DNA polymerase holds the template and non-template strands apart and Ribonucleotides (NTPs) enter the active site and base pair with the template strand.

A. Prokaryotic Transcription (following is an example of bacterial transcription, of E.coli):
The RNA polymerase holoenzyme contains 5 subunits: α2ββ'ωσ. The catalyic core α2ββ'ω is readily separated from the sigma subunit σ. Holoenzyme = core (α2ββ'ω) + σ
Initiation: The RNA polymerase binds to a DNA sequence called promoter found 10 and 35 nucleotides before (to the 5' end) of the transcription start site. The sequences vary but consensus sequence in many E.coli genes is
                                             -35      TTGACAT                                      -10 TATAAT (TATA box)
5'UTR (UnTranslatedRegion);    Purine-triphosphate (at position 1);   ATG: first translated codon of gene
The effect of sigma factor (from in vitro expts using either holoenzyme or catalytic core (no sigma σ)).
   Alone the catalytic core can synthesize RNA from a DNA template but in a random fashion. However the sigma factor ensures that
- only ATP or GTP are used in initiation site,
- and that binding and initiation only at promoters,
- and that it uses the correct template strand
So sigma factor gives specificity to promoter. Different sigma factors 'swhich on' genes with different promoters. Sigma is released after formation of the first phosphodiester bond.
Elongation: Core RNA polymerase. Nucleotides are added to the new RNA chain
Termination: Two mechanisms: intrinsic (non-specific or Rho independent) and Rho dependent
- Features of Intrinsic termination sites: G:C rich then an A:T rich region before the termination site. G:C rich region includes diadic symmetry (palindrome). Forms a hairpin loop. RNA's end in U6-U8. Associated with pausing of the RNA polymerase. Mutations in this region eliminate the pause.
Weak interaction between newly synthesized U rich RNA and DNA pauses transcription. RNA polymerase tries to backtrack to reform RNA-DNA hybrid but RNA hairpin too strong. Hiarpin loop is stabilised by hydrogen bonding between C and G nucleotides.
In most cases mRNA in bacteria is polycistronic (i.e. a single mRNA transcript is transcribed from a group of adgacent genes like an operon). Therefore, the mRNA encodes more than one protein. A few mRNAs code for one protein only and these are termed monocistronic. The mRNA is highly unstable and once translated is degraded in minutes. In fact, the 5' end starts to decay even before 3' end has been synthesized or even fully translated.   Summary video
   - Rho (ρ) dependent termination sites: Rho hexamer, 6 x 50000 doesn't bind to DNA, binds to RNA. Has ATPase activity. Model of action of ρ factor: Rho binds to RNA at rut (Rho utilisation) site and moves along RNA, 5'>3', by hydrolysing ATP. Reaches an RNA pol pauses at a rho dependent termination site. Not usually hairpin, 40-60 nucleotides, and is rich in C and poor in G. Interaction with the RNA pol. leads to the release of the RNA strand.
  
B. Eukaryotic transcription:
Video overview 
Transcription and Processing - nice
Differences from prokaryotic transcription: Most eukaryotes have many more genes than bacteria (few thousands to tens of thousands more). They also have large amounts of non-coding DNA (9 genes/million base pairs in humans vs 900 genes/million base pairs in bacteria). DNA in chromatin can affect the access of enzymes. 3 RNA polymerase enzymes in eukaryotes (what is he saying? 5 RNA pol enzymes): where RNA pol II transcribes mRNA. The process also involves additional control with enhancers, activators and mediators - this allows differential expression. In addition, RNA undergoes considerable processing before leaving the nucleus.
Eukaryotic promoters have upstream enhancer, then abit closer(downstream) to coding gene the upstream activator elements (GGGCGG, the GC box and GGCCATCT, the CAAT box -80) and abit closer the TATA box -25. Eukaryotic promoters have 2 or 3 TATA box, GC box and CAAT box.
Pyruvate dehydrogenase promoters: E1a

  {{ In Eukaryotes there is much greater amount of genetic information than prokaryotes. DNA complexed Histones and other proteins = Chromatin. Genetic information on many chromosomes. Process of transcription spatially/temporally separate from translation (occurs in nucleus. mRNA transcript process/cleaves and realigned before transport to cytoplasm. Many transcript never leave nucleus. During growth and development of organism differentiation and changes in gene expression often influence by cell interactions and external signals (e.g. hormones). RNApol of eukaryotes are larger and more complex each consists of 2large sub-units + 10-15 smaller ones. The polymerases consist of a common core of subunits. They are unable to bind directly to DNA but need accessory proteins to initiate transcription - called Transcription Factor. Most known about = RNA Pol II, transcribes all mRNA in Eukaryote. 

Pol I - tandemly repeated rRNA genes. For 18s, 5.8s, 28s rRNAs. In nucleolus.

Pol II - mRNA i.e. all protein genes, most small nuclear RNAs (snRNPs). In nucleoplasm

Pol III - 5s RNA, tRNA and cytoplasmic RNAS all of which are 300bp long, encode RNA with structural roles (i.e. do not code for protein) and repeated 10^3 times per genomes. In nucleoplasm}}

Initiation
   Eukaryotic RNA pol cannot bind to DNA. Prior to binding of RNA pol II (for mRNA), general transcription factors (GTFs) first bind to promoter site. Promoter site is located ~25bp downstream (5' end) of start point. TATA binding protein (TBP), initially binds the TATA promoter sequence called the TATA box part of TFIID. TBP also contains other subunits called TBP associated factors (TAFs) to make the TFIID. TFIID attracts other GTFs and RNA pol II to the form pre-initiation complex. TFIIH opens up the DNA helix at the start point (helicase) and also phosphorylates the end of part RNA pol II called the carboxyl tail domain. Enzyme is changing its shape so that it is released from complex to elongate RNA chain. The pre-initiation complex consists of these two components: RNA polII - core enzyme and GTFs.
  Chromatin structure needs to be changed to allow genes to be transcribed. Antirepressor TFs remodel/change chromatin in 2 ways: by binding to ATP hydrolysis-dependent remodelling complex and by histone modification.
Regulation of gene transcription: 
- The biological properties of a cell are mainly due to active proteins expressed in it. At any one time only a fraction of genes in genome are being transcribed and translated. Gene regulation occurs at many levels such as transcription, stability of mRNA, translation and post translational modification.
- Regulation is due to molecular signals received either in or ouside of the cell and cause binding of regulatory proteins to specific DNA sites close to protein coding region. Binding modulates rate of transcription by: activators - assisting RNA pol to bind to the promoter and repressors - preventing RNApol to bind to the promoter.
Transcription factors have structural domains that bind DNA (homeodomain binds major groove, zinc fingers bind minor groove)
  Prokaryotic control: E.coli contains 4.6 million nucleotide pairs and encodes for 4300 different proteins and only few proteins are expressed at a given time. Gene expression can depend on food source - induction i.e. Lac operon  (video 2 lac operon) .              CAP and Lac I repressor regulate the lac operon. Lactose binds to the lactose repressor, preventing it from binding. When lactose is absent, the repressor binds to the lac operator and transcription is OFF. In low glucose, available lactose conditions, CAP binds to the CAP operator, enhancing transcription, which is STRONGLY ON. In high glucose, available lactose conditions, CAP does not bind the CAP operator. However, the lac repressor is not bound, so there is WEAK transcription. The lac operon responds to low glucose levels in the cell by allowing it to metabolize lactose instead, if lactose is present
   Eukaryotic control: More complex because there are far more genes than in bacteria and extra variety when genes need to be turned on: fatal haemoglobin, pubertal hormones, seasonal growth changes, viral infections...Most genes are turned off (silenced) at any given time. Chromatin is not packed uniformly in a chromosome. Euchromatin: less densely packed/condensed. Heterochromatin: most condensed. Euchromatin contains more genes and is more expressed. The chemical modification of chromatin also affects expression. Locus Control Regions (LCR): genetic switches. In DNA control region, protein binds to LCR; enhancer=on, insulator=off.
       Enhancers (vs Promoters): overall, found LCR. They are more complex, larger (50-150bp), new/different sequence motifs - similar in many (homologous). They have distinct and redundant sub-elements (individual elements can be destroyed => no effect: duplication of one region makes up for mutation in another). They can work close to a promoter to 10^3 bp away either upstream or downstream and can even work when within a gene (=internal control region) or beyond 3' end of a gene (in some genes, e.g Ig can be in INTRON)
          Insulin acts through 3 main pathways to bring about multiple effects: PLCγ, Pl-3 kinase pathway (glucose uptake and enzyme activation (for glycogen synthesis)) and Ras-MAPK pathway (gene transcription and cell growth). 
             Pathway downstream of Ras:  Activated ras binds to and activates a serine kinase protein called raf triggering a series of serine kinase activations: Firstly, MEK, which in turn activates MAP-kinase (mitogen activated protein kinase) that moves to the nucleus and activates transcription factors. TFs known as mitogen activated proteins (include myc, fos and jun). These TFs cause the effects of the growth factors within cell -> stimulating protein synthesis, the cell cycle or differentiation of the cell.

Important info:

  RNA is a single stranded molecule and the RNA bases are exposed and available for interaction with othr RNA or DNA molecules. Contain the pentose sugar ribose. The 4 bases are Adenine (A), Guanine (G), Cytosine (C), and Uracil (U) that replaces Thymine (T) in DNA and they bond in a complementary manner. RNA is normally single stranded BUT the strand can folk back and bind on itself to form complicated structures; often a mix of single and double stranded regions (e.g. stem-loops, hairpin, pseudoknot, circular, etc.)

RNA vs DNA

   RNA                          DNA
Ribose                     Deoxyribose
A,C,G,U                   A,C,G,T
Single strand            Double strand
10 to 1000s                Millions

In nucleic acids the 9 nitrogen atom of purines and the 1 atom of pyrimidines  are bonded to the 1’ carbon atom of ribose/deoxyribose.

Only one methyl group different between U and T