Prokaryotes Gene Expression

Week 7th
S2-IPA Monday April 22nd 2014
S2-IPA Friday April 26th 2014
 Central Dogma of Biology
• The general flow of genetic information is

Any DNA copied to any single

stranded RNA molecule. There Process by which mRNA is
are three types of RNA molecules made into a functional protein
Difference between Prokaryotic and
Eukaryotic Genomes
 Difference between Prokaryotic and
Eukaryotic Genomes
Prokaryotes: Eukaryotes:
• Small genomes 0.5– • Large genomes 107–1010 bp
10·106 bp • Very low coding sequence
• High coding sequence density. Exons are scattered
density (i.e. in yeast in a vast sea of non-genic
genome, 75% is coding ‘noise’. (i.e. in human
sequences) genome, only ~3% are
• Introns are either non- coding seqs.)
existent or short. • Alternative splicing sites
 Prokaryotic Genomes

• Recall - prokaryotes have a single circular

chromosome
• Also - no cell nucleus, therefore no splicing out of
introns
• Therefore, prokaryotic gene structure is quite simple
Translational Translational
Promoter start site (AUG) stop site
region

Open Reading Frame

Transcriptional Operator Transcriptional

start site sequence stop site
What do Prokaryotic Genes look like?
5’ 3’

Open Reading Frame

Promoter region (maybe)

Ribosome binding site (maybe)

Termination sequence (maybe)

Start codon / Stop Codon

 In prokaryotes, RNA polymerase binds to the -10
and -35 regions of the promoter relative to the start
site of transcription (+1)

promoter operator
 Prokaryotic Gene Expression

Promoter Cistron1 Cistron2 CistronN Terminator

Transcription RNA Polymerase

mRNA 5’ 3’

1 2 N
Ribosome, tRNAs,
Translation Protein Factors
N N
N C
C
C
1 2 3
Polypeptides
Slide modified from:
http://biology.uky.edu/520/Lecture/lect8/lect8Notes.ppt
Prokaryotic gene expression
 Gene Expression
• There are four major events that occur
during the process of gene expression
– Transcription
– RNA processing (esp. in eukaryotes)
– Translation
– Protein processing
A Gene is a Transcription Unit
Promoter Terminator
Regulatory
sequence

DNA

Transcription

mRNA 5 3

Many
Start codons Stop
codon codon

Ribosomal-
binding site
 Prokaryotic Gene Structure

Shine-
Shine- Stop Codon Delgarno
Delgarno TAA, TAG,
+1 box ATG box Stop Codon
TGA ATG
Cis-Regulatory
DNA Elements Coding Sequence = ORF 1 Coding Sequence = ORF 2

USE/Promoter 5’ UTR = Leader Protein A Spacer = 5’UTR Protein B

/Operator sequence of 2nd cistron

Cistron 1 Cistron 2

Regulatory Sequences Structural or Coding Sequences Termination

sequence

Regulatory and Coding Sequence Unit = Operon

 Prokaryotic Gene Structure
Shine-Delgarno
Shine-Delgarno Stop Codon Stop Codon
box
+1 box ATG TAA, TAG, TGA ATG TAA, TAG, TGA
Cis-Regulatory
DNA Elements Coding Sequence= ORF Coding Sequence= ORF

USE/Promoter Protein A Protein B Terminator

5’ UTR = Leader Spacer = 5’UTR
/Operator sequence sequence
of 2nd cistron

Cistron 1 Cistron 2

Transcritption
5’ UTR = Leader
sequence Spacer

Polycistronic
ORF Protein A
mRNA ORF Protein B
+1 AUG AUG
Shine-Delgarno Stop Codon Stop Codon
Shine-Delgarno
box UAA, UAG, UGA UAA, UAG, UGA
box
Transcription Proceeds Through Three Steps
Initiation

• Transcription factors & RNA

polymerase recognize & bind the
promoter

• DNA adjacent to the promoter is

denatured forming the open
promoter complex

Elongation

• RNA polymerase moves along the

DNA in synthesizing a RNA transcript.
Synthesis is 5’3’ – Only 1 strand of
DNA is read as a template.

Termination

• A termination signal is reached

causing RNA polymerase to
dissociated from the DNA
Figure 12.2
 A Prokaryotic Promoter
(Pribnow Box= TATA Box)
Coding strand Transcriptional
Promoter start site
–35 sequence –10 sequence
+1
5 3
T TG AC A T AT AAT A
A AC T G T ATAT TA T
3 5
A
Template strand
5 3
RNA
Transcription
 Reaching A Consensus
–35 region –10 region +1 Transcribed
lac operon TTTACA N17 TATGTT N6 A

lacI GCGCAA N17 CATGAT N7 A

trp operon TTGACA N17 TTAACT N7 A

rrnX TTGTCT N16 TAATAT N7 A

recA TTGATA N16 TATAAT N7 A

lexA TTCCAA N17 TATACT N6 A

tRNAtyr TTTACA N16 TATGAT N7 A

Consensus TTGACA TATAAT

 Promoter Elements
• Gene expression begins with transcription
– RNA copy of a gene made by an RNA polymerase
– Prokaryotic RNA polymerases are assemblies of
several different proteins
• b’ protein binds to DNA template
• b protein links nucleotides
• a protein holds subunits together
• s protein recognizes specific nucleotide
sequences of promoters
 Promoter Elements

• b’, b and a often very similar from one

bacterial species to another
• s can vary (less well conserved)
– Several variants often found in a cell
– The ability to use several different s factors allows
a cell to turn on or off expression of whole sets of
genes
• For example, s32 turns on gene expressions for genes
associated with heat shock while s54 does the same for
nitrogen stress and genes that always need to be
expressed are transcribed by polymerases with s70
 Promoter Elements
• Each s factor recognizes a particular sequence
of nucleotides upstream from the gene
– s70 looks for -35 sequence TTGACA and -10
sequence TATAAT
– Other s factors look for other -35 and -10
sequences
• The match need not always be exact
• The better the match, the more likely transcription will
be initiated
 Promoter Elements
• Protein products from some genes are always
used in tandem with those from some other
genes
– These related genes may share a single promoter in
prokaryotic genomes and be arranged in an operon
– When one gene is transcribed, so are all of the others
- one polycistronic RNA molecule is produced
– The lactose operon contains three genes involved in
metabolism of the sugar lactose in bacterial cells
 RNA Polymerases

Structure of a bacterial Structure of a eukaryotic

RNA polymerase RNA polymerase II
 RNA Polymerases
• Differences between eukaryotes & prokaryotes
• Prokaryotes
– One enzyme with four subunits
• 2 α’s, 1 b, & 1 b’
• actual polymerase function
– Sigma factors (σ )
• recognize & bind promoter DNA sequence
• Eukaryotes
– 3 separate holoenzymes – each has ~12 subunits
• RNA Pol I – 28S, 18S, 5.8S rRNA
• RNA Pol II – mRNA, snRNA
• RNA Pol III – tRNA, 5S rRNA
– 3 sets of basal transcription factors
• recognize promoter DNA sequences
 The Process of Transcription
• Initiation
– Where/when most regulation of gene expression
occurs
– Different between prokaryotes & eukaryotes
• Elongation
– Essentially same between prokaryotes &
eukaryotes
– Some regulation, more in prokaryotes than
eukaryotes
• Termination
– Different between proks & euks
– Some regulation
Prokaryotic Transcription Initiation

Figure 12.6
 Rho Dependent Termination in Prokaryotes

rho utilization site

Rho protein is a
helicase

Figure 12.8 r-dependent termination

Rho Dependent Termination in Prokaryotes

Figure 12.8 r-dependent termination

 Rho Independent Termination in Prokaryotes
• r-independent termination requires two sequences in the RNA
– A stem-loop structure upstream of 7-9 U residues

NusA
Stabilizes the
RNA pol
pausing

Figure 12.9 r-independent termination

• During transcription, the gene determines the
sequence of bases along the length of an mRNA
molecule
DNA Gene 2
molecule
Gene 1

Gene 3

DNA strand 3 5
(template) A C C A A A C C G A G T

TRANSCRIPTION

U G G U U U G G C U C A
mRNA 5 3
Codon
TRANSLATION

Protein Trp Phe Gly Ser

Figure 17.4 Amino acid