transcription of microbes

Slide 1: 

TRANSCRIPTION

Slide 2: 

Synthesis of an RNA chain on a DNA template with the help of enzyme RNA polymerase. First step of Gene Expression Gene expression means how the information contained in genes is converted to molecules that determines the metabolism, structure and form of organism. TRANSCRIPTION

Slide 3: 

The Prokaryotic Transcription Need to understand the following: Differences between DNA and RNA. Differences and similarities between transcription and replication.

Slide 4: 

DNA vs RNA RNA differs from DNA in 3 ways: RNA is single stranded RNA has the sugar ribose RNA has the nitrogen base uracil instead of thymine.

Slide 5: 

Transcription/ Replication Similarities Polarity:- 5’-3’ Template Phosphodiester bond formation Phases:- Initiation Elongation Termination Polarity (5’ phosphate, 3’ hydroxyl) 5’ 3’

: 

Differences between replication and transcription Selective process

Slide 7: 

Components of transcription A. DNA template B. Raw materials (substrates) C. Enzyme involved

Slide 8: 

Template Single strand of DNA double helix Experimental evidence:-Oscar Miller, .Barbara Hamkalo and Charles Thomas(1970)-used e- microscopy to examine cellular contents and prove DNA act as a template for RNA synthesis. In cell, they saw Christmas tree structures which has thin central fibres(trunk of tree) to which were attached strings(branches) with granules Christmas tree

Slide 9: 

Christmas tree Observation:- DNA RNA Each “Christmas tree” represented a gene undergoing transcription. Transcription of each gene begins at the top of tree, little of DNA has been transcribed and RNA branches are short. As the transcription apparatus moves down the tree, transcribing more of the template produces long branches at the bottom Conclusion:- DNA is template used for RNA synthesis.

Slide 10: 

Template strand The strand which is used for transcription . It’s sequences are complementary to transcribed RNA. It is also known as antisense strand. Non template strand The strand of ds DNA other than template strand. It’s sequences are similar to transcribed RNA. (T/U) It is also known as sense or coding strand. vs

Slide 11: 

Only one strand of ds DNA serves as a template Experimental evidence:- Julius Marmur et al.,(1963) Bacteriophage SP8 (which infect Bacillus subtilis )- ds DNA. The two strands have different base composition and so different densities which permits the separation of two strands by equilibrium density gradient centrifugation into heavy and light DNA strands. Cont.....

Slide 12: 

Bacillus subtilis in a medium containing radiolabelled RNA precursor Bacteriophage SP8 Radioactively labelled RNA complementary to phage DNA Labelled RNA isolated Bacteriophage SP8 culture DNA isolated Heavy strand Light strand Hybridized Non Hybridized Conclusion:- RNA transcribed from one DNA strand (here H. Strand).

Slide 13: 

Cont..... In SP8, all the genes are transcribed from the same strand (also in most organisms) but different genes may be transcribed from different strands. e.g. adenovirus Organization of coding information in the adenovirus genome

Slide 14: 

Transcription unit:- A piece of DNA that encodes a single RNA molecule along with sequences necessary for its transcription It may includes critically three units Promoter RNA coding sequence Terminator No base assigned no. zero.

Slide 15: 

Promoters Promoters typically consist of a 40 bp region on the 5’ side of the transcription start site. It is the key point for the transcription control. RNA polymerase bind with promoter to initiate transcription. How protein recognize specific promoter sequence? These RNAP require specific sequences in DNA which came to know by comparing the sequences of different promoter (conserved sequences). DNA sequences that are usually recognized by transcription apparatus and required for transcription to take place.

Slide 16: 

Consensus sequences :- Sequences that possess considerable similarity or consensus. These represent probabilities . Or Fig: Promoter Capital letters denote bases found in those positions in more than 50% of promoters examined; small letters denote bases found in those positions in 50% or fewer of promoters examined. Bases that appear with highest frequency at each position when a series of seqs. believed to have common function are compared.

Slide 17: 

Four (some time five) conserved features in a bacterial promoter Start point -10 sequence -35 sequence Spacer region UP elements

Slide 18: 

Consensus Sequence

Slide 19: 

Start point The start point is the point where first cistron becomes available as soon as the 5 ′end of the mRNA is synthesized. Usually a purine (up to 90%). Purine is common central base in sequence CAT (Not highly conserved). -10 sequence (pribnow box) 6-7 bp region situated upstream to promoter. It’s consensus is 5’ -TATAAT-3. Probabilities T80A95T45A60A90T96 (out of 100). unwinding domain

Slide 20: 

-35 sequences 6 bp region situated upstream to promoter. It’s consensus is 5’-TTGACA-3. Probabilities T82T84G78A65C54A45 (out of 100). It’s combination with -10 is referred as core promoter elements. Recognition domain Spacer the -35 to -10 sequences 16 or 18 bp (90% of promoters ) Spacers that are longer or shorter than the consensus length make weak promoters

Slide 21: 

UP element (Upstream promoter) Additional element other than core p. elements. Situated upstream to core promoter element (-40 to -60). It’s work in stress condition for production of large amount of rRNA (E.coli). Fig: The rrnB P1 promoter .

Slide 22: 

UP element is a true promoter element- because recognized by RNAP itself It stimulates transcription of rrnBP1 gene by a factor of 30 in the presence of RNAP alone. Fis sites:- 3 in no. position- -60 to -150 Not actual promoter element Binds by folded C terminal domain of α subunit

Slide 23: 

Role of mutation in promoter up mutations Which increase the rate of transcription by - Increase homology with one of the consensus sequence Bring the distance b/w c. seq. closer to 17 bp. down mutations Which decrease the rate of transcription- Decrease homology with one of the consensus sequence. Make distance between them more or less from 17 bp. -35 seq Reduce the rate of closed complex formation. -10 seq. Slow the rate of conversion of closed to open form.

Slide 24: 

Fig: The -35 sequence is used for initial recognition, and the -10 sequence is used for the melting reaction that converts a closed complex to an open complex

Slide 25: 

There is a gap over several bands This shows that RNAP protected certain bonds from cleavage by DNAase. Mapping promoters Technique used for this is DNA footprinting.

Slide 26: 

2) RNA coding sequence Seq. of DNA ntds that is copied into an RNA molecule 3) Terminator Seq. of ntds that signal where transcription is to end. B) Raw material:- ribonucleotide (r NTPs) c) Enzyme RNA Polymerase

Slide 27: 

RNA Polymerase As early as, 1960-61-RNAPs were discovered in animals, plants and bacteria. By 1969, the polypeptides that makeup the E.coli polymerase had been identified by SDS-PAGE. In Bacteria (simple system) - all three classes of RNA are transcribed by the same RNA polymerase. In Eukaryotes (complex system) - each class is transcribed by a different RNA Polymerase RNAP I - rRNAs RNAP II - mRNAs RNAP III - tRNAs & small ribosomal RNAs (RNAP for short)

Slide 28: 

Model system Phage T7 RNAP Small RNAP comprising single polypeptide chain of <100kD. Recognize a few promoter on phage DNA. Model system for characterizing the binding of RNAP to DNA and initiation reaction. Rate- ~200 ntds/sec at 37O C. Structure- similar to DNA polymerase DNA lies in a “palm” surrounded by a “fingers” and a “thumb”. Cont….

Slide 29: 

Recognizes its target sequences by binding to the bases in major groove at a position upstream from the start point. Uses a specificity loop formed by β ribbon(not in DNAP) DNA polymerase

Slide 30: 

Common to all RNAPs- recognize specific bases in DNA that are upstream to the start point. During initiation-enzyme conformation remains same and active site can hold a transcript of 6-9 ntds. Transition from initiation to elongation-when enzyme begins to move along DNA. Nacent transcript extends beyond active site and interacts with specificity loop. RNA emerges to surface of enzyme when12-14 ntds are synthesized.

Slide 31: 

Bacterial RNA Polymerase In E.coli, RNA polymerase is a large multimeric (many polypeptide chains) 465 kD complex. Around 7000 RNAP is in E.coli cell out of which 2000-5000 are involved in transcription with the no. generally depends upon the growth condition. a a2 a2b a2bb’ = core enzyme CORE ENZYME Sequence-independent, nonspecific transcription initiation SIGMA SUBUNIT interchangeable, promoter recognition + Holoenzyme a2bb’σ (Greek letter-specificity)

Slide 32: 

Eubacterial RNA polymerases

Slide 33: 

Evidence for promoter recognition function E.coli with modified - Subunit Infected with T4 phage Reduced affinity for promoter recognition by holoenzyme a-subunit b and b’ seqs. are related to those of largest subunit of eukaryotic RNAP. b’ binds mainly DNAs while b to NTPs . Mutation in rpoB and in rpoC affects all the stages of transcription.

Slide 34: 

Many bacteria have multiple types of σ factors. Each type of σ initiate the binding of RNAP to a particular set of promoter. σ 70 , is used for general transcription, other sigma factors are induced by particular environmental conditions. (A number in the name of a factor indicates its mass.). (σD instead of F in Bacillus subtilis)

Slide 35: 

Shape of enzyme with and without σ factor (How polymerase binds to DNA) Model of the interaction between E. coli RNA polymerase holoenzyme and a promoter. (a) Core (red) with its closed channel (25Å)binds to σ (blue) to form holoenzyme with an open channel. (b) Holoenzyme binds loosely to DNA (green) to form a closed complex. (c) Holoenzyme grasps the DNA tightly, forming an open promoter complex. (d) Finally, σ dissociates, leaving core firmly clamped around the DNA, so it can transcribe the DNA processively.

Slide 36: 

Scan DNA and identify promoters Bind to promoters Initiate transcription Elongate the RNA chain Terminate transcription Be responsive to regulatory proteins (activators and repressors) RNA Polymerase Has Many Functions Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the  subunit of RNAP and inhibits the RNA synthesis (Initiation) .

Slide 37: 

Transcription process Initiation phase: RNA-pol recognizes the promoter and starts the transcription. Elongation phase: the RNA strand is continuously growing. Termination phase: the RNA-pol stops synthesis and the nascent RNA is separated from the DNA template.

Slide 38: 

INITIATION Until 1980, common assumption that initiation ended when RNAP formed the first phosphodiester bond, joining the first two ntds in the growing RNA chain. Disproved by:-Agamenon Carpousis and Jay Garalla Reported more complex initiation. Several very small oligontds (2-6bp) formed without leaving the promoter before actual RNA synthesis i.e. Abortive transcript. Other researchers found 9 or 10 ntd transcripts also.

Slide 39: 

(a) The polymerase holoenzyme binds nonspecifically to the DNA. (b) The holoenzyme slides along the DNA, searching for a promoter. (c) The holoenzyme has found a promoter and has bound loosely, forming a closed promoter complex. (d) The holoenzyme has bound tightly, melting a local region of DNA and forming an open promoter complex INITIATION .

Slide 40: 

E). The polymerase incorporates the first nine or ten nt into the nascent RNA f) Promoter clearance :- The polymerase clears the promoter, loses its σ factor, and begins the elongation phase. e) f)

Slide 41: 

First ntd incorporated in RNA retains all the 3 of its phosphate while others only α. Transcription stars with purine –ATP more often than GTP. Initiation is rate limiting step. In O.P. complex-RNAP extends from position -44 to +3. Melting region is 17±1 bp of DNA to form a transcription bubble and the bubble of this size moves along with RNA synthesis. No primer requirement. Cont….

: 

b. Elongation The release of the  subunit causes the conformational change of the core enzyme. The core enzyme slides on the DNA template toward the 3 end. Free NTPs are added sequentially to the 3 -OH of the nascent RNA strand. RNA-pol, DNA segment of ~40nt and the nascent RNA form a complex called the transcription bubble. The 3 segment of the nascent RNA hybridizes with the DNA template, and its 5 end extends out the transcription bubble as the synthesis is processing.

: 

Transcription bubble Negative supercoiling Positive supercoiling

Slide 44: 

c. Termination When RNAP reaches a terminator at the end of a gene, it falls off the template, releasing the RNA Termination:- 1) Intrinsic (rho independent) 2) rho dependent

-independent termination : 

-independent termination Two structural features A hairpin in the secondary structure: – G-C rich region near the base of stem U residue rich region (near 3’ end of RNA). Stem-loop structure alters the conformation of RNAP leading to the pause of the RNAP moving. Among all the base pairing, rU-dA is the most unstable.

Slide 46: 

-independent termination

Slide 47: 

-dependent termination Termination require Rho factor. Rho factor- -terminator protein that binds to a rut site on nacent RNA. -hexameric ATP dependent helicase, Rut site –rich in C residue ,poor in G and no sec. structure.

Slide 48: 

Antitermination

Slide 49: 

Phage lamda has 2 antitermination proteins- pN and pQ that act on different transcription units . It is used at two stages of phage expression. The site for antiterminator protein is in upstream of the terminator site in transcription unit.