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