Chapter Overview
RNA polymerases and sigma factors
● Transcription: DNA is converted to RNA
● The genetic code, ribosomes, and tRNAs
● Translation: RNA is converted to protein
● Bioinformatics: Mining the genomes
●
1
Introduction
The cell accesses its vast store of data in its
genome by:
- Reading a DNA template to make an
RNA copy (transcription)
- And decoding the RNA to assemble
protein (translation)
After translation, each polypeptide is
properly folded and placed at the correct
cellular or extracellular location.
2
RNA Polymerase
Is a complex enzyme that carries out transcription by
making RNA copies (called transcripts) of a DNA
template strand
In bacteria, the RNA pol holoenzyme is composed of:
- Core polymerase: a2, b, b´
- Required for the elongation phase
-Holoenzyme: s , a2, b, b´
- Sigma factor: s
- Required for the initiation phase
3
RNA Polymerase
•
RNA poymerase links nucleotides in the 5’  3’
•
Opens DNA by itself (helicase is not required)
•
Transcription is slower than replication (~ 50
nucleotides/sec)
•
Lacks proofreading function (errors 10-4).
4
Figure 8.2
Figure 8.3
5
The sigma factor helps the core enzyme
detect the promoter, which signals the
beginning of the gene.
Every cell has a “housekeeping” sigma factor.
- In E. coli, it is sigma-70.
- Recognizes consensus sequences
at the –10 and –35 positions, relative to
the start of the RNA transcript (+1)
A single bacterial species can make several
different sigma factors.
6
How Sigma Factor Recognizes
Specific DNA Sequences
Orientation of the promoter determines the
direction of the transcription
7
Alignment of sigma -70 (s) dependent promoters from
various genes is used to generate consensus sequences.
Yellow= conserved region; Brown= transcript start site.
8
Transcription of DNA to RNA
Transcription occurs in three phases:
1) Initiation: RNA pol holoenzyme binds to
the promoter
- The closed RNA pol complex becomes
open.
2) Elongation: The RNA chain is extended
3) Termination: RNA pol detaches from the
DNA, after the transcript is made
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10
Transcription Initiation
•
Transcription can occur either strands
•
Only one DNA strand is transcribed
(sense strand)
•
Transcription proceeds 5’  3’
•
The first base is usually a purine (A or
G) added to the +1 site.
•
Orientation of the promoter determines
the direction of the transcription
11
Energy released in this process is used to
build phosphodiesterase bonds
12
Transcription Elongation
• Is the sequential addition of ribonucleotides
•
•
•
from nucleoside triphosphates
The original RNA polymerase continues to
move along the template, synthesizing
RNA at ~ 45 bases/sec.
The unwinding of DNA ahead of the moving
complex forms a 17-bp transcription
bubble.
Positive supercoils ahead are removed by
DNA topoisomerases.
13
Now we have some idea of how RNA
polymerase recognizes the beginning of a
gene and how the transcription proceeds!
But how does it know when to stop
14
The secret is in the sequence !
15
Transcription Termination
There are two types of transcription:
- Rho-dependent
- Relies on a protein called Rho and a
strong pause site at the 3´ end of the gene
- Rho-independent
- Requires a GC-rich region of RNA, as well
as 4–8 consecutive U residues
16
Figure 8.8
17
Termination of transcription
DNA
5’
Promoter Operator
A
Transcription
B
Terminator
C
3’
18
Antibiotics that Affect
Transcription
Rifamycin B
- Selectively binds to the bacterial RNA pol
- Inhibits transcription initiation
Actinomycin D
- Nonselectively binds to DNA
- Inhibits transcription elongation
19
Six Classes of RNA
Messenger RNA (mRNA): Encodes proteins
Ribosomal RNA (rRNA): Forms ribosomes
Transfer RNA (tRNA): Shuttles amino acids
Small RNA (sRNA): Regulates transcription
or translation
tmRNA: Frees ribosomes stuck on damaged
mRNA
Catalytic RNA: Carries out enzymatic
reactions
20
Translation: mRNA  Protein
mRNA contains codes for how to make
a proteins !
21
The Genetic Code
Consists of nucleotide triplets called codons
There are 64 possible codons:
- 61 specify amino acids.
- Include the start codons (AUG)
- 3 are stop codons (UAA, UAG, UGA)
The code is degenerate or redundant.
- Multiple codons can encode same amino acid.
The code operates universally across species.
- Remarkably, with very few exceptions
22
Figure 8.11
23
The Genetic Code
• Degeneracy: redundancy (e.g.
leucine has 6 codons and alanine
has 4 codon)
24
tRNA Molecules
Are decoder molecules that convert the language
of RNA into that of proteins
tRNAs are shaped like a clover leaf (in 2-D) and a
boomerang (in 3-D).
A tRNA molecule has two functional regions:
- Anticodon: Hydrogen bonds with the mRNA
codon specifying an amino acid
- 3´ (acceptor) end: binds the amino acid
25
Figure 8.12B
-About 60 different t-RNAs in
bacteria
-About 20 aminoacyl-tRNA
synthetases
Figure 8.13
26
Figure 8.15
The charging of
tRNAs is
carried out by a
set of enzymes
called
aminoacyltRNA
synthetases.
27
The Ribosome
• Ribosomes are composed of two subunits,
each of which includes rRNA and proteins.
• In prokaryotes, the subunits are 30S and
50S and combine to form the 70S ribosome.
• The 30S contains 21 proteins (S1-S21)
assembled around 16S rRNA
• The 50S contains 31 proteins (L1-L31)
associated with 5S and 23 S rRNA
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29
The 70S ribosome harbors three binding sites
for tRNA:
- A (acceptor) site: Binds incoming
aminoacyl-tRNA
- P (peptidyl-tRNA) site: Harbors the tRNA
with the growing polypeptide chain
- E (exit) site: Binds a tRNA recently
stripped of its polypeptide
30
Translation of RNA to Protein
Polypeptide synthesis occurs in 3 phases:
1) Initiation: which brings the two ribosomal
subunits together, placing the first amino acid in
position
2) Elongation: which sequentially adds amino
acids as directed by mRNA transcript
3) Termination: which releases the completed
protein and recycles ribosomal subunits
Each phase requires a number of protein
factors and energy in the form of GTP.
31
How do ribosomes find the
right Reading Frame?
32
Defining a Gene
Alignment of a bacterial structural gene with
its mRNA transcript
Figure 8.21
33
Open Reading Frames (ORF)
mRNA sequence
AUG GCA UUG CCU UAG
Start -------------------------Stop
Reading Frame # 1  AUG GCA UUG CCU
met ala leu pro
Reading Frame # 2  A UGG CAU UGC CU
try his cys
Reading Frame # 3  AU GGC AUU GCC U
gly Ile ala
34
Translation Initiation
Figure 8.23
35
Translation Elongation`
Three steps are repeated:
• t-RNA-carrying an amino acid
binds to “A” site
• peptide bond formation occurs
• the message must move by one
codon
36
Translation Termination
37
38
Coupled transcription
and translation
in prokaryotes.
39
Antibiotics that Affect
Translation
Streptomycin: Inhibits 70S ribosome formation
Tetracycline: Inhibits aminoacyl-tRNA binding
to the A site
Chloramphenicol: Inhibits peptidyltransferase
Puromycin: Triggers peptidyltransferase
prematurely
Erythromycin: Causes abortive translocation
Fusidic acid: Prevents translocation
40
Protein Modification
Protein structure may be modified after
translation:
- N-formyl group may be removed by
methionine deformylase.
- The entire methionine may be removed by
methionyl aminopeptidase.
- Acetyl groups or AMP can be attached.
- Proteolytic cleavages may activate or
inactivate a protein.
41
What is bioinformatics?
Bioinformatics is the field of science in which
biology, computer science, and information
technology merge to form a single discipline.
The ultimate goal of the field is to enable the
discovery of new biological insights as well as
to create a global perspective from which
unifying principles in biology can be discerned
42
Bioinformatics
Since 1998, the complete genomes of more than
225 microbial species have been published.
This wealth of information has spawned a new
discipline called bioinformatics, which is
dedicated to comparing genes of different species.
Data from bioinformatics enable scientists to make
predictions about an organism’s physiology and
evolutionary development.
- Even without culturing the organism in a lab
43
Annotating the Genome Sequence
Annotation of the DNA sequence is basically
understanding what the sequence means.
- It requires computers that look for patterns, such
as regulatory sequences, open-reading frames
(ORFs), and rDNA and tRNA genes
An ORF is a sequence of DNA that encodes
an actual polypeptide.
- In eukaryotes, finding ORFs is complicated by
the presence of introns.
44
DNA Sequence
>A01_TK-M13F-Plate5.ab1 1360
0 1360 ABI
TTCCTAAGCTGGTTACTAGACTGCACATTGGGCCCTCTAGAGATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATT
CGCCCTTGTGCCAGCCGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGG
CTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAG
ATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAGCACCGGTGGCGAAGGCGGATCTCTGGGCCGATACT
GACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTG
TGGGCCACATTCCACGTGGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTC
AAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGA
CATACACCGGAAACATTCAGAGATGGGTGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGT
GAGATGTTGGGTTAAGTCCCACAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGG
GAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCT
ACAATGGCCGGTACCATGAGCTTCCATACCGCAAGGTGGAGCGAAACTCAAAAAGCCGGTCTCACTTCCGATTGGGGTCTCC
ACCTCCCCCCCCTGCAATTTGATCCCGTGTAATACTGGATATAAGTGTTGCGGGGAAACCTTCCCGGGGGTGTTTACCCCCCC
CTTCAAGAGGGAATTCCTCCCAACCGGCGGCGCCTTTCTAGTGAGAACCCACCCGTGTGCCAACCTTTGATTAATTTATGGGG
GGTTGTTTTTTTTATTAACAAAGNNNNNGTNACANNGGNNAANCGCCCCGGGGCCGTTCACCCCCCCTATAATTGCCCTTTGTT
GACGAATTACCCCCCTTTTCGCCCGTGGTCCGCGACCCCAAATACCCCACAAGCAGGTCCCAGCCCACCCAATTCCCCCATG
TCCCCCCCCATCCCCCTCGTCTTCTTAACCTTCGCGCCGAGTGGTGTTAAACAGGGGAGGTCCGCGCTGGATATCGTTTTTTT
TGATGTTATGGCAGCTCCTCCTAGATTTATAGACGCCCCCCGCG
45
Predicting Open Reading Frames (ORFs) in a
DNA sequence
Predicting a Open Reading Frame (ORF). Prediction begins locating the:
-Start codon: AUG in m-RNA (TAC on sense DNA).
-Stop codons: UAA, UAG, and UGA in m-RNA (ATT, ATC, and ACT on
sense DNA)
-Ribosome-binding site: upstream of start the start codon
46
Mycoplasma mycoides: Color code indicates gene clustering by
function. The inner most circle shows GC content. Red , > 50%
and black, < 50%.
47
Evolutionary Relationships
Genes that are homologous likely evolved
from a common ancestral gene.
- Orthologous genes
- Genes duplicated via appearance of a new
species
- Have identical function in different organisms
- Paralogous genes
- Genes duplicated within a species
- Have slightly different tasks in a cell
48
49
Bioinformatics
Many computer programs and resources used
to analyze DNA and protein sequences are
freely available on the Web.
- BLAST
- Multiple Sequence Alignment
- KEGG
- Motif Search
- ExPASy
- Joint Genome Institute
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