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Transcription in Prokaryotes M.Prasad Naidu MSc 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 Gene: Structure of signals Gene 2 Gene 1 • 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
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 bubbleor 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.
Initiation Sigma dissociates
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 Sequences • In 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 site • RNA transcript at the terminating site is self-complementary • The 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 terminationsite String of Us Intrastrand complementary bases
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 E. 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:
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 ntis 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-dependent: • 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
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 Termination site Initiation site RNA fibrils
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
