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A gene
is a segment of DNA used to make a functional product– either an RNA or a polypeptide
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Transcription
is the first step in gene expression, literally means the act or processof making a copy, the term refers to copying a DNA sequence into an RNA sequence
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The central dogma of genetics
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Promoter
site for RNA polymerase binding; signals the beginning of transcription.
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Regulatory sequences
site for binding of regulatory proteins that influence the rate of transcription. Regulatory sequences can be found in a variety of locations.
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Ribosome-binding site
translation begins near this site in the mRNA. In eukaryotes,the ribosome scans them RNA for a start codon.
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The sequence of codons
in the mRNA determines the sequence of amino acidsin a polypeptide.
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Bacterial mRNA may be polycistronic,
which means it encodes two or more polypeptides
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The DNA strand that is transcribed
is the template strand
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The RNA transcript is complementary
to the template strand
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The opposite strand
is the coding strand or the sense strand as well as the non-template strand
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The base sequence
is identical to the RNA transcript except for the substitution of uracilin RNA for thymine in DNA
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Transcription factors
recognize the promoter and regulatory sequences to control transcription
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Transcription
occurs in three stages:Initiation Elongation Termination
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Initiation
The promoter functions as a recognition site for transcription factors. The transcription factor(s) enables RNA polymerase to bind to the promoter. Following binding, theDNA is denatured into a bubble known as the open complex.
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Elongation/synthesis of the RNA transcript
RNA polymerase slides along the DNA in an opencomplex to synthesize RNA.
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Termination
A terminator is reached that causes RNA polymerase and the RNA transcript to dissociate from the DNA.
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Over 90% of all genes are structural genes
which are transcribed into mRNA Final functional products are polypeptides
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The RNA transcripts from nonstructural genes
are not translated
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Promoters(Bacteria)
DNA sequences that “promote” gene expression they direct the exact location for the initiation of transcription typically located just upstream of the site where transcription of a gene actually begins bases in a promoter sequence are numbered in relation to the transcription start site
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System of promoters(bacteria)
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RNA polymerase catalyzes the synthesis of RNA(Bacteria)
RNA polymerase holoenzyme is composed of:Core enzyme Sigma factor These subunits play distinct functional roles
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Initiation of Bacterial Transcription
The RNA polymerase holoenzyme binds loosely tothe DNAIt then scans along the DNA, until it encounters apromoter regionWhen it does, the sigma factor recognizes boththe –35 and –10 regionsA region within the sigma factor that contains ahelix-turn-helix structure is involved in a tighterbinding to the DNA
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End of Initiation (Bacteria)
The binding of the RNA polymerase to the promoterforms the closed complexThen, the open complex is formed when theTATAAT box in the -10 region is unwoundA short RNA strand is made within the opencomplexThe sigma factor is released at this point
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Elongation in Bacterial Transcription
The core enzyme slides down the DNA to synthesize an RNA strand The DNA strand used as a template for RNAsynthesis is termed the template or antisense strandThe opposite DNA strand is called the coding strand
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Last phase of elongation
Behind the open complex, the DNA rewinds back into a double helix
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Termination of Bacterial
- TranscriptionTermination is the end of RNA synthesisTermination occurs
- when the short RNA-DNAhybrid of the open complex is forced toseparate
- releasing the newly made RNA as wellas the RNA polymerase
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E. coli has two different mechanisms for termination
– rho-dependent termination• Requires a protein known as r (rho)– rho-independent termination• Does not require r (rho)
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TRANSCRIPTION IN EUKARYOTES
the basic features very similar to bacteria• gene transcription in eukaryotes is more complex• more complex cells (organelles)• higher cellular complexity means more genes encoding proteins are needed • Multicellularity adds another level of regulation–express genes only in the correct cells at the proper time
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Nuclear DNA is transcribed by three different RNA polymerases (eukaryotes)
- RNA pol I
- RNA pol II
- RNA pol III
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RNA pol I (eukaryotes)
Transcribes all rRNA genes (except for the 5S rRNA)
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RNA pol II (eukaryotes)
Transcribes all structural genes synthesizes all mRNAs Transcribes some snRNA genes
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RNA pol III (eukaryotes)
Transcribes all tRNA genes and the 5S rRNA gene
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Features of most promoters (for structural genes)(eukaryotes)
Regulatory elements TATA box Transcriptional start siteS
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Core promoter (eukaryotes)
consists of the “TATA box” and “transcriptional start site”• determining the start point for transcription– The core promoter by itself produces a low level of transcription or basal transcription
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Regulatory elements(eukaryotes)
short DNA sequences that affect the binding of RNA polymerase to the promoter
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Transcription factors(eukaryotes)
bind to these regulatory elements and influence the rate of transcription
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Three categories of proteins are required for basa ltranscription to occur at the promoter(eukaryotes)
RNA polymerase II Five different proteins called general transcription factors (GTFs) A protein complex called mediator
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RNA Pol II transcriptional termination (eukaryotes)
• Pre-mRNAs are modified by cleavage near their 3’end with subsequent attachment of a string of adenines• Transcription terminates 500 to 2000 nucleotides downstream from the polyA signal
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RNA MODIFICATION (eukaryotes)
The sequence of DNA in the coding strand corresponds to the sequence of nucleotides in the mRNA• The sequence of codons in the mRNA provides the instructions for the sequence of amino acids in the polypeptide• eukaryotic structural genes reveal that they are not always colinear with their functional mRNAs
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Transcription produces the entire gene product(eukaryotes)
– Introns are later removed or excised– Exons are connected together or spliced
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RNA transcripts can be modified in several ways (eukaryotes)
For example• Trimming of rRNA and tRNA transcripts• 5’ Capping and 3’ polyA tailing of mRNA transcripts
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Exonucleases
cleave a covalent bond between two nucleotides at one end of a strand
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Endonucleases
can cleave bonds within a strand
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The translation
of the mRNA codons into amino acid sequences leads to the synthesis of proteins
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Genes that encode polypeptides
are termed structural genes These are transcribed into messenger RNA (mRNA)
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Archibald Garrod
First to propose (at the beginning of the 20th century) a relationship between genes and protein production Garrod studied patients who had defects in their ability to metabolize certain compounds
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Alkaptonuria propose
He proposed that a relationship exists between the inheritance of the trait and the inheritance of a defective enzyme He described the disease as an inborn error of metabolism
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Genetic code
Translation relies on the genetic code The genetic information is coded within mRNA in groups of three nucleotides known as codons
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AUG (specifies for methionine) =
start codon
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UAA, UAG and UGA =
termination, or stop, codons
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Overview of gene expression
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Polypeptide synthesis
has a directionality that parallels the 5’ to 3’ orientation of mRNA
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20 amino acids
that may be found in polypeptides
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Nonpolar amino acids are
hydrophobic
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Polar and charged amino acids are
hydrophilic
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There are four levels of structure in proteins
Primary Secondary Tertiary Quaternary
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A protein’s primary structure
is its amino acid sequence
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The primary structure of a protein folds to form regular, repeating shapes known as
secondary structures
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There are two types of secondary structures
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Tertiary structure
The short regions of secondary structure in a protein fold into a three-dimensional tertiary structure This is the final conformation of proteins that are composed of a single polypeptide Structure determined by hydrophobic and ionic interactions as well as hydrogen bonds and Van der Waals interactions
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Quaternary structure
Proteins made up of two or more polypeptides have aquaternary structure This is formed when the various polypeptides associate with one another to make a functional protein
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Functions of proteins
A category of proteins are enzymes Accelerate chemical reactions within a cell Can be divided into two main categories Anabolic enzymes Synthesize molecules and macromolecules Catabolic enzymes Break down large molecules into small ones Important in generating cellular energy
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Structure and function of tRNA
tRNAs play a direct role in the recognition of codons in the mRNA In particular, the hypothesis proposed that tRNAhas two functions 1. Recognizing a 3-base codon in mRNA 2. Carrying an amino acid that is specific for that codon
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During mRNA-tRNA recognition,
the anticodonin tRNA binds to a complementary codon in mRNA
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The secondary structure of tRNAs exhibits
a cloverleaf pattern It contains Three stem-loop structures A few variable sites An acceptor stem with a 3’ single strand region
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The enzymes that attach amino acids to tRNAsare known as
aminoacyl-tRNA synthetases
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Aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different molecules
Amino acid, tRNA and ATP (for energy)
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The aminoacyl-tRNA synthetases are responsible for the
“second genetic code”
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Translation occurs on the surface of a large macromolecular complex termed
the ribosome
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Bacterial cells have one type of ribosome
Found in their cytoplasm
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Eukaryotic cells have two types of ribosomes
One type is found in the cytoplasm The other is found in organelles Mitochondria ; Chloroplasts
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A ribosome is composed of structures called
the large and small subunits
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Ribosomes contain three discrete sites
Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site)
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Translation initiaton
The mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex This process requires three Initiation Factors The initiator tRNA recognizes the start codon in mRNA In bacteria, this tRNA is designated tRNAfmet It carries a methionine that has been covalently modified to N-formylmethionine The start codon is AUG
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The binding of mRNA to the 30S subunit is facilitated by
a ribosomal-binding site or Shine-Dalgarno sequence This is complementary to a sequence in the 16S rRNA
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Translation elongation
During this stage, amino acids are added to the polypeptide chain, one at a time
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Translation termination
The final stage occurs when a stop codon is reached in the mRNA In most species there are three stop or nonsense codons UAG UAA UGA These codons are not recognized by tRNAs, but by proteins called release factors Indeed, the 3-D structure of release factors mimicsthat of tRNAs
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Bacteria have
three release factors
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Eukaryotes only have one
release factor eRF, which recognizes all three stop codons
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Bacterial Translation Can Begin Before Transcription Is Completed
Bacteria lack a nucleus Therefore, both transcription and translation occur in the cytoplasm
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Transcription + translation
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Gene regulation
refers to the ability of cells to control their level of gene expression
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Constitutive genes
are unregulated and have constant levels of expression
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Benefits of gene regulation
Conserves energyproteins produced only when neededAccurate gene expressionEnsures genes expressed in appropriate cell type and at the correct stage in development
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Gene regulation in prokaryotes
used to respond to changes in the environment
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Gene regulation in eukaryotes
All of the organism’s cells contain the same genome but express different proteomes
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Bacterial gene regulation
Most commonly occurs at the level of transcription Or control rate mRNA translated Or regulated at protein or post-translation level
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Eukaryotic gene regulation
Transcriptional regulation commonRNA processingTranslationPost-translation
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Bacterial gene regulation
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Eukaryotic gene regulation
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Transcriptional regulation in bacteria
Involves regulatory transcription factors Bind to DNA near a promoter and affect transcription of one or more nearby genes Repressors inhibit transcriptionNegative control Activators increase the rate of transcriptionPositive control
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Operon
in bacteria is a cluster of genes under transcriptional control of one promoterRegulatory region called operator
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lac operon
In E. coli contains several genes for lactose metabolism lacP - promoter 3 structural geneslacZ – β-galactosidase Allolactose important in lac operon regulationlacY – lactose permeaselacA – galactosidase transacetylase
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When lactose is absent
Lac repressor binds to nucleotides of lacoperator site preventing RNA polymerase from transcribing lacZ, lacY and lacARNA polymerase can not move forward
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When lactose is present
Allolactose is a small effector moleculeallolactose molecules binding to lacrepressor prevents repressor from bindingProcess called induction and lac operon is inducible
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Operon is turned off
when CAP is not bound
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CAP binding to DNA
enhances RNA polymerase binding which increases transcription
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trp operon
In E. coli, encodes enzymes required to make amino acid tryptophan Regulated by a repressor protein encoded by trpR gene Binding of repressor to trp operator site inhibits transcription
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DNA methylation
DNA methylase attaches methyl groups Common in some eukaryotes but not all In mammals, 5% of DNA is methylated Usually inhibits transcription Unmethylated areas are correlated with active genes
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