TRANSCRIPTION IN PROKARYOTES: (Intro- part 1)

Important points:

• RNA polymerase initiate transcription de novo without primers.
• Multiple RNA Polymerase can transcribe the same gene at same time. Thus, a cell can synthesize large number of transcripts from single gene in short time.
• DNA replication is more accurate because DNA is the molecule in which genetic material is stored and by replication it is passed on. Any mistake that arises during replication can therefore easily be catastrophic. It becomes permanent in the genome of that individual and also get passed on subsequent generations.
• Transcription, in contrast, produces only transient copies and normally several from each transcribed region. Thus, a mistake during transcription will rarely do harm than render one out of many transient transcripts defective.

RNA Polymerase ;

In prokaryotes (bacteria):

• Only a single type of RNA Polymerase.
• Alone can synthesize all types of RNA.
• The core enzyme comprises of :- 2 copies of alpha-subunits = alpha' and alpha "(homologous to RPB3 and RPB11), 1 each copy of beta, beta', omega subunits (homologous to RPB1, RPB2 and RPB6).

In eubacteria or eukaryotic cell:

• Three types of RNA pol. are there which synthesize different mRNA:
• RNA Pol I = pre ribosomal RNA, 18S RNA, 28 RNA, 5.8s RNA
• RNA Pol II = pre mRNA, snRNA, LINE sequences, miRNA
• RNA Pol III = tRNA, 5srRNA.

Plants have Pol IV and Pol V.

Shape of RNA Pol. resembles crab claw in which two largest subunits beta' and beta (or RPB1 and RPB2) forms the pincers. And the active center cleft contains two Mg2+ ions.

TRANSCRIPTIONAL UNIT:

Sequence of nucleotides in DNA that codes for a single RNA molecule, along with the sequences necessary for its transcription; normally contains promoters, RNA-coding sequences and terminator.

+1 is the site from where transcription starts. Bases under +1 are called downstream and outside the Transcription unit or before start point are called upstream elements.

 PROMOTER is the DNA sequence that initially binds the RNA Pol. together with other factors and initiates the process. 

• They also determine the rate of initiation. 
• They are cis acting elements (present near the gene).
• They can be in upstream or downstream elements.
• They can be strong or weak sequences.
• Promoters with sequences closer to the consensus sequences are generally stronger.
• By strength means, how many transcripts it initiates in a given time and it is influenced by how well - a) promoter binds the polymerase initially b)how efficiently it supports isomerization (closed to open complex)  c) how ideally polymerase can escape.

CONSENSUS SEQUENCES:

These are the conserved sequences derived by aligning number of (300) sequences known to function as promoter.

In case of prokaryotes, there are two consensus sequences:

1. Present at -10 position (contain six base pair AT rich region) (5'- TATAAT-3'). This sequence is present at non-template or sense strand.

2. Present at -35 position ( TTGACA). 

These two sequences are centered and are present at upstream region. And are separated by a non-specific stretch of 17-19 nucleotides.

Promoter region contain four different parts:

• Consensus sequences (-10, -35)
• Upstream (UP) elements
• -10 extended elements
• Discriminator region (before -10)

SIGMA FACTOR:

RNA Pol. as a core enzyme combine with the sigma factor or subunit to form holoenzyme.

sigma 70 can be divided into four regions: 

• Region 4  = Two helices form common DNA-binding motif called helix-turn-helix. First helix inserts in major groove and interact with bases of -35 region. -35 provides binding energy to secure Pol. to promoter. Second helix lies top of groove, making contact with DNA backbone. E.g., transcriptional activator and repressor in bacterial cells.
• Region 2 = Interact with -10 region by alpha-helix. Alpha helix contains several essential aromatic amino acids that interact with bases on non-template strand and stabilize melted DNA. -10 is within that element that DNA melting is initiated. This region is bound to single stranded -10 element and also of the entire intact open complex. Two bases of non-template flipped out and inserted into pocket within sigma protein where they stabilize the unwound state of promoter.
• Region 3 = Extended -10 element is recognized by alpha helix in region 3. Interact with two specific base pair of this element.
• Region 1.2  recognizes the discriminator region.
• UP elements are recognized by carboxyl terminal domain of sigma called alpha-CTD. While the alpha-NTD is embedded in the body of enzyme.

Sigma 2 and 4 region are separated by 75 A when sigma is bound in holoenzyme. This is same between centers of -10 and -35. This distance is provided by region 3.2. Therefore, it is known as Linker DNA or sigma 3/4.