Transcript Transcription and Translation - Microbiology and Molecular Genetics

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).
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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.
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How Sigma Factor Recognizes
Specific DNA Sequences
Orientation of the promoter determines the
direction of the transcription
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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
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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
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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
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Figure 8.8
17
Termination of transcription
DNA
5’
Promoter Operator
A
Transcription
B
Terminator
C
3’
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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
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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
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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)
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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.
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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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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
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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.
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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
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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
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Translation Termination
37
38
Coupled transcription
and translation
in prokaryotes.
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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
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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.
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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
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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
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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.
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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%.
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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
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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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