1 chapter overview ● rna polymerases and sigma factors ● transcription: dna is converted to rna...
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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
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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.
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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: 2, , ´
- Required for the elongation phase
-Holoenzyme: 2, , ´
- Sigma factor: - Required for the initiation phase
RNA Polymerase
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• 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).
RNA Polymerase
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Figure 8.3
Figure 8.2
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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.
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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
Transcription of DNA to RNA
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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
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• 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.
Transcription Elongation
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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 !
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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
Transcription Termination
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Figure 8.8
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DNA Promoter AOperator B C Terminator
5’ 3’
Termination of transcription
Transcription
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Rifamycin B
- Selectively binds to the bacterial RNA pol
- Inhibits transcription initiation
Actinomycin D
- Nonselectively binds to DNA
- Inhibits transcription elongation
Antibiotics that Affect Transcription
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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
Six Classes of RNA
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Translation: mRNA Protein
mRNA contains codes for how to make a proteins !
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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
The Genetic Code
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Figure 8.11
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The Genetic Code
• Degeneracy: redundancy (e.g. leucine has 6 codons and alanine has 4 codon)
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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
tRNA Molecules
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Figure 8.12B
Figure 8.13
-About 60 different t-RNAs in bacteria
-About 20 aminoacyl-tRNA synthetases
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The charging of tRNAs is carried out by a set of enzymes called aminoacyl-tRNA synthetases.
Figure 8.15
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• 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
The Ribosome
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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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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.
Translation of RNA to Protein
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How do ribosomes find the right Reading Frame?
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Alignment of a bacterial structural gene with its mRNA transcript
Defining a Gene
Figure 8.21
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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 CUtry his cys
Reading Frame # 3 AU GGC AUU GCC Ugly Ile ala
Open Reading Frames (ORF)
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Translation Initiation
Figure 8.23
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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
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Coupled transcription and translation in prokaryotes.
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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
Antibiotics that Affect Translation
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Protein ModificationProtein 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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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
What is bioinformatics?
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BioinformaticsSince 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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>A01_TK-M13F-Plate5.ab1 1360 0 1360 ABITTCCTAAGCTGGTTACTAGACTGCACATTGGGCCCTCTAGAGATGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGCCCTTGTGCCAGCCGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAGCACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCCACATTCCACGTGGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAACATTCAGAGATGGGTGCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCACAACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGGGACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACCATGAGCTTCCATACCGCAAGGTGGAGCGAAACTCAAAAAGCCGGTCTCACTTCCGATTGGGGTCTCCACCTCCCCCCCCTGCAATTTGATCCCGTGTAATACTGGATATAAGTGTTGCGGGGAAACCTTCCCGGGGGTGTTTACCCCCCCCTTCAAGAGGGAATTCCTCCCAACCGGCGGCGCCTTTCTAGTGAGAACCCACCCGTGTGCCAACCTTTGATTAATTTATGGGGGGTTGTTTTTTTTATTAACAAAGNNNNNGTNACANNGGNNAANCGCCCCGGGGCCGTTCACCCCCCCTATAATTGCCCTTTGTTGACGAATTACCCCCCTTTTCGCCCGTGGTCCGCGACCCCAAATACCCCACAAGCAGGTCCCAGCCCACCCAATTCCCCCATGTCCCCCCCCATCCCCCTCGTCTTCTTAACCTTCGCGCCGAGTGGTGTTAAACAGGGGAGGTCCGCGCTGGATATCGTTTTTTTTGATGTTATGGCAGCTCCTCCTAGATTTATAGACGCCCCCCGCG
DNA Sequence
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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
Predicting Open Reading Frames (ORFs) in a DNA sequence
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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