prokar transcription
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Transcription in Prokaryotes
M.Prasad NaiduMSc Medical Biochemistry, Ph.D,.
Gene expression begins with transcription
RNA copy of a gene made by an RNA polymerase
Prokaryotic RNA polymerases are assemblies of several different proteins
Bacterial genomes have simple gene structure
- Promoter
-35 sequence (T82T84G78A65C54A45) 15-20 bp
-10 sequence (T80A95T45A60A50T96) 5-9 bp (Pribnow Box)
- Start of transcription : initiation start: Purine90 (sometimes it’s the “A” in CAT)
- Translation binding site (Shine-Dalgarno) 10 bp upstream of AUG (AGGAGG)
- One or more Open Reading Frame
•start-codon (unless sequence is partial)
•until next in-frame stop codon on that strand ..
Separated by intercistronic sequences.
- Termination
Bacterial Gene: Structure of signals
Gene 1 Gene 2
RNA polymerase must know where the start of a gene is in order to copy it
RNA polymerase has weak interactions with the DNA unless it encounters a promoter A promoter is a specific sequence of
nucleotides that indicate the start site for RNA synthesis
General Steps of Transcription
Initiation: Binding of RNA polymerase to double stranded DNA Development of closed promoter complex Development of open promoter complex Start of transcription by adding the first two
ribonucleotides. Elongation: - Formation of transcription bubble or Transcription elongation complex.
Progression of the complex gradually in the 3’ direction to elongate the initiated RNA chain.
Rapid process: up to 40 nucleotides per second. On the same gene there are several RNA strands being
transcribed in a staggered fashion. Termination:
Terminator sequences signal stop of transcription.
Sigma dissociates
Initiation
Irreversible form of Open Complex Formation
Generation of Abortive Initiation Products
Transcription Bubble
RNA Elongation
Reads template 3’ to 5’ Adds nucleotides 5’ to 3’
(5’ phosphate to 3’ hydroxyl)
Synthesis is the same as the leading strand of DNA
Polymerization is polar: enzyme works by adding to a free 3’ hydroxyl in growing mRNA chain.
RNA Synthesis
RNA pol moves nt by nt, unwinds the DNA as it goes
Will stop when it encounters a STOP.
RNA pol leaves, releasing the RNA strand
Termination of Transcription
Factor-independent termination
Factor-dependent termination– 3 factors
• Rho (), Tau () and NusA– Rho best studied
Termination of transcription
RNA: single stranded nucleic acid• can form secondary structures
Rho-dependent termination: protein signal • Rho binds to RNA; able to cause RNA &
RNA polymerase to leave DNA→ termination
Rho-independent signal: hairpin or stem- loop RNA structure forms, followed by several uracils
→ termination
Terminator SequencesIn prokaryotes there are two types:
1. Intrinsic: Rho (ρ) independent terminator
Contains a G-C rich region followed by six or more A-T sequences. Causes the formation of a double stranded RNA
called a hairpin loop. Retards the movement of the RNA polymerase
along the DNA molecule, and causes termination at the A-T rich region.
2. Extrinsic: Rho-dependent terminator
Requires a protein factor called Rho (ρ).Rho protein trails the RNA polymerase until
it reaches a GC rich region, when Rho catches up with the polymerase.
Rho protein pulls off RNA from transcription bubble.
1. Rho-independent terminator siteRNA transcript at the terminating site is
self-complementaryThe bases can pair to form a hairpin
structure with a stem and loop, a structure favored by its high G-C content
The stable hairpin is followed by a sequence of 4 or more U residues
The RNA transcript ends within or just after them
Intrinsic termination site
String of Us
Intrastrand complementarybases
Mechanism of Rho-independent Termination RNA polymerase pauses when it encounters
such a hairpin formed at the terminator site The RNA-DNA hybrid helix produced after
the hairpin is unstable because of its content of rU-dA base pairs, the weakest of the four kinds of pairs
Nascent RNA is pulled off from the DNA template and then from the enzyme
DNA template strand now joins its partner to form the DNA duplex
Control of trp operon by attenuation:
stalled translation allows region 2 to interact with region 3
3 & 4 cannot interact
regions 3 & 4 interact; termination results
Global control systems in Global control systems in E. coliE. coli::
In global control systems: many genes, pathways regulated simultaneously in response to a specific environmental signal
• e.g., regulon: collection of genes and/or operons controlled by common regulatory protein
• Sporulation in Bacillus: another global control system:
2. Rho-dependent terminator
Rho (ρ) Protein
Rho is an RNA-dependent ATPase Also an RNA-DNA helicase It is an hexamer, with a mass of 275 kDa (each
subunit is of 46 kDa) It binds to ssRNA at Rut site – a stretch of 72 nt
is bound, 12 per subunit It is brought into action by sequence located in
the nascent RNA ATPase activity enables it to move
unidirectionally along the nascent RNA
Effect of rho protein on the size of RNA transcripts
Rho factor: factor mediated termination
In an ATP-mediated reaction, a rho protein complex binds to the mRNA and unwinds RNA from the DNA template
Recognition sites may not have hairpins or U tracts; tend to be C-rich
?
Rho-dependent:
One Transcriptional unit
Two contiguous genes
RNA is released so we can make many copies of the gene, usually before the first one is done Can have multiple RNA polymerase molecules on
a gene at a time
Initiationsite
Termination site
RNA fibrils
Summary
Types of mRNA
In Bacteria:
Monocistronic mRNA
Polycistronic mRNA
In Eukaryotes:
Monocistronic mRNA
Polycistronic mRNA
Many prokaryotic mRNAs are polycistronic Contain sequences specifying the synthesis of
several proteins
A polycistronic mRNA molecule possesses a series of start and stop codons
In case it codes for three proteins: Start, Protein1, Stop – Start, Protein2, Stop – Start, Protein3, Stop
Abou 5-20 bases may be present between one stop codon and the next start codon. These are called Spacers.
The segment of RNA corresponding to a DNA cistron is called a Reading frame
Polycistronic Polycistronic vsvs Monocistronic mRNA Monocistronic mRNA
spacers
Processing of pre-rRNA transcripts in E. coli
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