Transcription in Prokaryotes

Transcription: production of mRNA copy of the DNA gene.

Transcription

Eukaryote model

Not all RNA is translated into protein:

• Some RNA is structural - e.g. ribosomal RNA (rRNA)• Some RNA is functional - e.g. transfer RNA (tRNA)• Some RNA is chromosomal (some viruses)

The production of protein-encoding RNA in bacteria is the subject of this lecture.

RNA

Transcription

From which DNA strand is RNA synthesized?

Transcription usually takes place on only ONE of the DNA strands (though not necessarily the same strand throughout the entire chromosome).

Transcription

5'-GTCACCCATGGAGG-3' Nontemplate strand

3'-CAGTGGGTACCTCC-5' Template strand

5'-GUCACCCAUGGAGG-3' mRNA

RNA growth always in the 5' 3' direction

5'3'

3'5'

5' 5' 5'

5'3'

3' 3' 3'

mRNA

mRNA mRNA mRNA

DNADNA

Transcription

RNA Polymerase

The synthesis of RNA from a DNA template is carried out by enzymes known formally as DNA-dependent RNA polymerases, now simply referred to as RNA polymerases

Transcription

ACCCATGG C A5'-GT GG-3' Nontemplate “CODING”strand

3'-CA CC-5‘ Template strand G T TGGGTACC

CAUG C C A5'-GUC

RNA polymerase

Transcription

RNA polymerases have the following properties:

The enzymes are template dependent, requiring double-stranded DNA

The enzymes require the four nucleoside triphosphates (ATP, GTP, CTP, and UTP)

The enzymes copy (read) the template DNA strand in the 3' to 5' direction

The enzymes synthesize the RNA in the 5' to 3' direction

RNA Polymerase

Order of events in Transcription1) Binding of polymerases to the initiation site, the promoter.

Prokaryotic polymerases can recognise the promoter and bind to it directly.

2) Unwinding (melting) of the DNA double helix by a helicase. In prokaryotes the polymerase has the helicase activity.

3) Synthesis of RNA based on the sequence of the DNA template strand, using nucleoside triphosphates (NTPs) to construct RNA.

4) Termination of synthesis. NOTE: the “STOP” codon in the genetic code for the end of peptide synthesis is NOT the end of termination.

Transcription

Prokaryotic RNA Polymerase: Core Enzyme

Chain initiation and interaction with regulatory proteins

Catalytic center: chain initiation and elongation

DNA binding

RNA Polymerase

RNA Polymerase

The core enzyme has the ability to synthesize RNA, however, the initiation point of RNA synthesis is non-specific.

An additional subunit, the sigma factor, is required to initiate RNA synthesis at specific locations in the DNA, termed

the promoter.

RNA Polymerase

Prokaryotic RNA Polymerase: Holoenzyme

Promoter recognition

• For any given gene, RNA synthesis always starts at the same point on the DNA, the promoter. What is a promoter?

• Hypothesis: Because one RNA polymerase copies every gene and binds to the promoter in each gene to do so, the promoters in different genes must have similarities. Similarities in DNA must lie in the sequence of nucleotides so the promoters of every gene must have the same sequence of nucleotides.

Promoters

David Pribnow tested this by comparing the sequences in the promoter regions of five genes from E. coli. He found a conserved sequence of nucleotides in each. This was called the Pribnow box.

Pribnow

Promoters

• The Pribnow box lies 10 nucleotides from the transcription start point (TSP). A second was later found 80 nucleotides away.

-80 -10 TSPTTGACA TATAAT

DNA

Pribnow box

Promoters

5'3'

3'5'

RNA

• The sequences found in promoters are to some extent imaginary. Very few genes actually contain these sequences but they all contain a sequence that is only a few nucleotides different. The consensus sequence is a “best average”.

Consensus sequences

Promoters

GCGTTGTCATGC gene1 AATGTGACAGCT gene2 TGCTAGACACAG gene3 GAATTGAGAAAA gene4 CTTTTCACATTC gene5 AGCTAGACAGGG gene6 TCGTTGGCACCA gene7 CCAATGACCATT gene8 ATGTTGACTTGC gene9 TTGACA consensus not actually

in any of the genes

Promoters

• Just because consensus sequences have been found, this doesn’t mean that they are functional. What is the evidence that they actually work?

Promoters

• Although sequences can vary from the consensus, some mutations stop the promoter from working. In these cases, it demonstrates that the consensus sequence is a functional promoter.

• Genes that are transcribed strongly have sequences more like the ideal consensus than genes that are transcribed weakly.

Promoters

Transcription

Promoter region in DNARNA polymerase

RNA polymerase scans DNA double helix, searching for a promoter site.

(1) Sigma binds to promoter region.

Sigma residues Y425, Y430 and W434 directly involved in the unwinding (melting) of the double helix.

Initiation

(1) Sigma binds to promoter region, recognizing both the -35 and -10 regions. The resulting structure is termed a closed promoter complex.

The promoter is rich in A and T.  The AT pair involves two hydrogen

bonds whereas the CG pair involves three hydrogen bonds.  Therefore, AT pairs

are easier to separate.

Initiation

Initiation

(2) After the DNA strands have been separated at the promoter region by the helicase activity of the sigma subunit, forming an open promoter complex. The core subunit () can then start to synthesize RNA.

(3) Following initiation, the sigma subunit is released after approx. 10 ribonucleotides have been polymerized,

Elongation

Synthesis of the RNA strand continues until the core polymerase reaches the termination site.

Termination

In prokaryotes, the transcription is terminated by two major mechanisms:

Rho-independent (intrinsic) and Rho-dependent.

Termination

The Rho-independent termination signal is a stretch of 30-40 bp sequence, consisting of many GC residues followed by a series of T ("U" in the transcribed RNA). 

The resulting RNA transcript will form a stem-loop structure to terminate transcription

Rho-independent

The terminator has the following structure:

GC rich GC rich PolyA

GC rich GC rich PolyU

DNA

RNA

Complementary

Termination

U CU G G C A U C G C G C G G C C G C G

C

UAAUCCCACAG CAUUUU

GC rich regions

Poly U

RNA

stem-loop structure

Termination

TAG GC rich 1 GC rich 2 ATrich

RNA

RNA polymerase

As transcription proceeds, the two GC rich regions base pair. This leaves a short poly U rich region, which cannot pair strongly enough to hold the RNA onto the DNA. The polymerase comes off with it.

Termination

3 5

35

Rho-dependent.

In vitro, E. coli RNA polymerase holoenzyme transcribes DNA into a very long RNA.

The ability of the in vitro reaction to make natural length RNA is restored by the addition of a protein factor, called rho ().

RNA transcript length:

By holo polymerase in vitro

In vivo

By holo polymerase + rho

Termination

Termination

Analysis of termination sites dependent on rho revel a stem loop structure near the 3‘ end of the RNA, with NO U-rich tale.

Rho binds to RNA and can, if provided with ATP, move along the RNA.

Rho also has ATP-dependent helicase activity.

It has been established that six Rho proteins form a hexamer to terminate transcription, but the precise mechanism is not clear.

(1) The Rho hexamer first binds to the RNA transcript at an upstream site which is 70-80 nucleotides long and rich in C residues .

Model for rho termination

Termination

(2) Upon binding, the Rho hexamer moves along the RNA in the 5-3 direction, trying to catch up with the RNA polymerase.

(3) When the polymerase pauses, which happens when secondary structures form near the 3 end of the RNA, rho catches up and melts the

RNA-DNA duplex in the replication bubble, causing termination.

Termination

Protein

Ribosomes

DNA

RNA

Transcription and translation

Termination

DNA

UAG

Ribosome dissociates from the RNA when they encounter a stop codon.

Termination

DNA

UAG Rho factor binds to specific sites on naked RNA.

(i.e. RNA without ribosomes)

Termination

DNA

UAG

Termination

RNA polymerase pauses at stem loop, while rho moves along RNA, 5-3.

Termination

DNA

UAG

Rho catches up with polymerase, melting RNA-DNA duplex, causing polymerase to dissociate.

Termination of transcription can serve a role in regulating gene expression in prokaryotes

This is the subject of the final lecture in this series.

Termination

Suggested reading

Transcription (2000) In: An Introduction to Genetic Analysis. pp 300-306. Griffiths, A. J. F,. Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M. (Eds). Freeman and Company, New York.

http://www.nottingham.ac.uk/bennett-lab/lee.html