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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