Download 2.4 RNA and Protein Synthesis

Biotechnology: Genes and Genomes
Reviewing cells, protein synthesis, etc.
Chapter Contents
•2.1 A Review of Cell Structure
•2.2 The Molecule of Life
•2.3 Chromosome Structure, DNA
Replication, and Genomes
•2.4 RNA and Protein Synthesis
•2.5 Mutations: Causes and Consequences
•2.6
Revealing the Epigenome
© 2013 Pearson Education, Inc.
2.1 A Review of Cell Structure
•All cells have these structures:
•Plasma Membrane – double-layer structure
of lipids and proteins that surrounds its outer
surface
•Cytoplasm – inner contents of a cell
between the nucleus and plasma membrane
•Ribosomes – structures in the cytoplasm
which facilitate protein synthesis
•DNA – molecules which carry genetic code
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2.1 A Review of Cell Structure
•Comparison of Prokaryotic and
Eukaryotic Cells
Insert table 2.1
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2.1 A Review of Cell Structure
•Prokaryotic Cells
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2.1 A Review of Cell Structure
•Eukaryotic cells (include plant and animal
cells)
Insert figure 2.2
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2.1 A Review of Cell Structure
•Plasma membrane: fluid, dynamic, complex
double-layered barrier made of what
macromolecules?
–Roles include cell adhesion, cell-cell communication, cell
shape, transport molecules in and out of cell, is selective
barrier
•Cytosol: nutrient-rich, gel like fluid that makes up
cytoplasm
•Organelle: compartment where chemical rxns and
cell processes take place
–Each organelle has its own biochem rxns
–Allows coordination of chem rxns within a cell
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2.1 A Review of Cell Structure
Work in groups to complete the table
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2.1 A Review of Cell Structure
Work in groups to complete the table
© 2013 Pearson Education, Inc.
2.1 A Review of Cell Structure
Work in groups to complete the table
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2.2 The Molecule of Life
•Evidence that DNA is the Inherited Genetic
Material
–1869 Friedrich Miescher: "nuclein"
•Could not be broken down by proteases
•Based on this observation, would you consider it a
protein?
•Discovered nuclein had acidic properties: renamed "nucleic
acids"
•What are the 2 types of nucleic acids?
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2.2 The Molecule of Life
–More Evidence that DNA is the Inherited Genetic
Material
–1928 Frederick Griffith
•Two strains of bacteria (Streptococcus pneumoniae)
–Virulent disease causing smooth strain (S cells) and harmless rough
strain (R cells)
•S cells are surrounded by capsule (smooth coat)
•R cells lack the capsule
•Did experiment in which mice were injected with either a) S
strain; b) R strain; c) heat killed S strain or d) mix of heat
killed S strain and live R strain to see if mice would survive
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2.2 The Molecule of Life
•What are the macromolecules that make up the capsule?
•Why did they heat the S strain and not the R?
•Discuss the process of transformation in fig. d.
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2.2 The Molecule of Life
•Transformation: DNA is taken up by another
bacterium
•Heat treatment broke open S cells and their
capsule so DNA was released
•Living R cells took up S cell DNA which
transformed the property of R cells so they
became virulent/pathogenic
•Molecular biologists use transformation
routinely to introduce genes into bacteria for
cloning
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2.2 The Molecule of Life
•Definitive evidence that DNA Is the Inherited
Genetic Material
–1944 Oswald Avery, Colin MacLeod, and Maclyn McCarty
•Homogenized mixtures of bacterial cells from large batches of
Streptococcus pneumoniae
–Treated extracts with proteases; RNases; DNases
–Did transformation expts with the extracts
•Found that extracts from killed S cells treated with DNase mixed with living R
cells did not transform because DNA in S cells was degraded
•Experiment provided evidence that DNA is genetic material and proved
that it was the transforming factor in the Griffith experiments
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2.2 The Molecule of Life
•Follow-up questions related to Avery, MacLeod,
McCarty experiment
•Why did extracts treated with proteases still
maintain ability to be transformed?
•Why did extracts treated with RNases still
maintain ability to be transformed?
•Why did extracts treated with DNases lose the
ability to be transformed?
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2.2 The Molecule of Life
•DNA Structure
–Building block of DNA is the nucleotide
–Each nucleotide is composed of
•Pentose (5-carbon) sugar called deoxyribose
•Phosphate molecule
•A nitrogenous base
–The nitrogenous bases are the interchangeable
component of a nucleotide
•Each nucleotide contains one base
–Adenine (A), thymine (T), guanine (G) or cytosine (C)
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2.2 The Molecule of Life
•Building block of DNA = nucleotide
•What is the difference between deoxyribose and ribose
sugar?
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2.2 The Molecule of Life
•DNA Structure
–Rosalind Franklin and Maurice Wilkens provided xray crystallography data to show helical structure
–James Watson and Francis Crick used data from
Franklin and Wilkens; Chargaff base pairing rules to
develop wire model that revealed the definitive
structure of DNA
–"The Molecular Structure of Nucleic Acids: A
Structure for Deoxyribose Nucleic Acid" published in
Nature on April 25, 1953
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2.2 The Molecule of Life
•DNA Structure
–Nucleotides are joined together to form long strands of DNA
and each DNA molecule consists of two strands that join
together and wrap around each other to form a double helix
–Nucleotides in a strand are held together by
phosphodiester bonds
–What part of a nucleotide and adjacent nucleotide does
the phosphodiester bond connect?
–Each strand has a polarity – a 5' end and a 3' end
–Polarity refers to the carbons on what part of a
nucleotide?
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2.2 The Molecule of Life
•DNA Structure
–The two strands of a DNA molecule are held
together by hydrogen bonds
•Formed between complementary base pairs
•Adenine (A) pairs with thymine (T)
•Guanine (G) pairs with cytosine (C)
–The two strands are antiparallel because their
polarity is reversed relative to each other
–DNA resembles a twisted ladder
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2.2 The Molecule of Life
•What are the rungs of the ladder?
•What is the backbone or sides of the ladder?
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2.2 The Molecule of Life
•What is a gene?
•Sequence of nucleotides that provides cells with
instructions to synthesize a protein or type of RNA
•On average genes are 1000–4000 nucleotides long
•Genes influence how cells, tissues and organs
appear
–Define the term trait:
–*not all genes are used to produce a protein
•(example) State the function of genes involved in making tRNA.
© 2013 Pearson Education, Inc.
2.3 Chromosome Structure, DNA
Replication, and Genomes
•Chromosome Structure
–Chromosomes – where cells package DNA
–Chromatin – strings of DNA and DNA-binding
proteins called histones
State of DNA inside the nucleus when the cell
is NOT dividing - long, thin, chromatin.
•During cell division chromatin is coiled into fibers that wrap
around each other so chromosomes are highly coiled
•Why would chromatin be condensed during cell division
based on what you know about the number of bases in
every cell?
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Most human cells have two sets (pairs) of 23
chromosomes (paternal and maternal), or 46
chromosomes total
These are called homologous pairs
–Autosomes – chromosomes 1-22
–Sex chromosomes – chromosome pair # 23
•X and Y chromosomes
•Gametes (sex cells) contain a single set of 23
chromosomes (haploid number, n)
•All other cell in body are somatic cells
•What type of cell is a kidney cell?
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Characteristics common to eukaryotic chromosomes
•Chromosome consists of two thin, rodlike structures of DNA
called sister chromatids
–Exact replicas of each other copied during DNA replication
–During cell division, each sister chromatid is separated
–Each chromosome has a single centromere – region consisting of
intertwined DNA and protein that join sister chromatids together and also
contain proteins that attach chromosomes to microtubules
•Centromere delineates each sister chromatid into 2 arms – p and q
•Each arm of chromosome ends with a telomere- highly conserved
repetitive nucleotide sequence that attach chromosomes to nuclear
envelope
–telomeres allow cells to divide without losing genes) but during aging
and cancer progression telomeres become shortened
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Chromosome Organization
•What is a potential consequence of shortening of telomeres during
the aging process?
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Karyotype- way to study chromosome number and basic
aspects of chromosome structure
–Spread cells on microscope
slide and treat with chemicals
to release and stain
chromosomes
–Chromosomes can be
aligned and paired based
on staining pattern and size
•Based on the karyotype, which chromosome is the largest? Explain
how you came up with your answer (work in groups).
•What would a karyotype for Trisomy 21 (Down Syndrome) look
like? Explain.
© 2013 Pearson Education, Inc.
2.3 Chromosome Structure, DNA
Replication, and Genomes
•DNA Replication
–Somatic cells divide by a process called mitosis
–Mitosis
•One cell divides to form two daughter cells, each with an
identical copy of the parent cell DNA
•A single skin cell would produce how many cells and
how many chromosomes/cell?
•In order to accomplish this, the DNA of the parent cell must
be copied prior to mitosis
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•DNA replication
•Sex cells divide by a process called meiosis – parent cell
divides to create 4 daughter cells which can be sperm or egg
cells
•The DNA in each daughter cell is not an identical copy of the
parent cell
•Chromosome number is cut in half to the haploid number
•How many sets of chromosomes does each daughter
cell have?
•Through sexual reproduction a fertilized egg called zygote is
formed and zygote divides by mitosis to form an
embryo = complete set of 46 chromosomes
© 2013 Pearson Education, Inc.
2.3 Chromosome Structure, DNA
Replication, and Genomes
•Semiconservative Replication
–DNA replication occurs in such a manner that, after
replication, each helix contains one original (parental) DNA
strand and one newly synthesized DNA strand
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Steps in DNA Replication
1.Unwinding the DNA
–Helicase enzyme breaks the hydrogen bonds between complimentary
base pairs that hold the two DNA strands together; "unzips" DNA
–Single strand DNA binding proteins bind to each strand and prevent
them from base pairing and reforming a double helix
–Separation of strands occurs in regions called origins of replication
2.Adding short segments of DNA 10–15 nucleotides long
called DNA primers
–Primase enzyme synthesizes DNA primers
–DNA primers start the replication process because they serve as binding
sites for DNA Polymerase – enzyme that synthesizes new strands of
DNA
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2.3 Chromosome Structure, DNA
Replication, and Genomes
•Steps in DNA Replication
3.Copying the DNA
–DNA polymerase enzyme binds to the
DNA primers
–Uses nucleotides to synthesize complementary
strands of DNA
–Always works in one direction – 5' to 3' direction
–What kind of bond is formed between the
phosphate in one nucleotide and sugar in the
previous nucleotide?
© 2013 Pearson Education, Inc.
2.3 Chromosome Structure, DNA
Replication, and Genomes
•Steps in DNA replication
•Since DNA Pol only goes in 5'–3' direction,
replication along leading strand is continuous and is
discontinuous along lagging strand
•Why is it discontinuous on lagging strand?
•Short Okazaki fragments are synthesized as DNA
Pol works on lagging strand
•DNA primers are replaced with DNA nucleotides
using DNA Pol
•Covalent bonds are formed between Okazaki
fragments with DNA ligase
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2.3 Chromosome Structure, DNA
Replication, and Genomes
© 2013 Pearson Education, Inc.
2.3 Chromosome Structure, DNA
Replication, and Genomes
•Genome – all of DNA in organism's cell
•DNA contains instructions for life in form of
genes
•Human genome has 20,000 genes scattered
among 3 billion base pairs of DNA!
•What is the study of genomics?
•What was the purpose of the human
genome project that was completed in
2003?
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CENTRAL DOGMA:
http://www.wiley.com//legacy/college/boyer/04700037
90/animations/central_dogma/central_dogma.htm
2.4 RNA and Protein Synthesis
•Transcription – genes are copied
(transcribed) from DNA code into
RNA code
•Translation – RNA code (exact
copies of genes) is read into a
protein sequence of amino acids
•Through production of RNA and protein synthesis DNA controls
properties of the cell and its traits
•Give an example of a trait using the words in the above figure.
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•Transcription
–Occurs only in segments of chromosomes that contain
genes
–RNA polymerase unwinds DNA helix and copies one
strand of DNA into RNA
•Binds to a promoter region – consensus nucleotide sequence
•Transcription factor (TF) proteins are DNA binding proteins that bind to
specific regions on DNA – purpose of transcription factors is to help RNA
Pol find the promoter
–TF can act to speed up transcription (like Red Bull drink) or stop
transcription (like Tylenol PM)
•Enhancers are DNA nucleotides that also play role in transcription
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2.4 RNA and Protein Synthesis
•Transcription
•After RNA Pol binds to promoter, it unwinds a region of DNA to separate
the 2 strands
•The template strand is copied by RNA Pol
•Copies template DNA in a 5' to 3' direction into RNA
•Uses nucleotides
–Adenine, uracil, guanine, and cytosine
–A-U, C-G
–What kind of covalent bond is formed between ribonucleotides?
–At end of gene, RNA polymerase encounters the termination
sequence to create loops at the end of RNA so RNA polymerase and
newly formed strand of RNA are released from DNA molecule
–RNA strand is called a messenger RNA (mRNA)
–Multiple copies of mRNA are transcribed from each gene during
transcription
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2.4 RNA and Protein Synthesis
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2.4 RNA and Protein Synthesis
•mRNA is not the only type of RNA that gets
transcribed.
•Name 2 other types of RNA that are produced
by transcription.
•Do these 2 types of RNA carry information that
directly codes for synthesis of protein? Work in
groups and explain.
•New class of non-protein coding RNA: microRNA
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•mRNA Processing
–Initial mRNA produced is the primary transcript
•Immature and not fully functional
–Three modifications before primary transcripts are ready for
protein synthesis (takes place in nucleus)
•RNA splicing – splice out the DNA not coding for proteins (introns) and
retain the protein coding sequence of the gene (exons)
–Alternative splicing – multiple proteins produced from single gene
•3' PolyA tail – 100–300 adenine nucleotides added to protect mRNA
from RNA degrading enzymes; increase its stability and availability for
translation
•Addition of a 5' cap – guanine base containing methyl group allows
ribosome recognition
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2.4 RNA and Protein Synthesis
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2.4 RNA and Protein Synthesis
TRANSLATION
•Components of translation – in cytoplasm
•mRNA – messenger RNA: copy of gene (acts as
messenger by carrying genetic code from nucleus to
cytoplasm where info is read into protein)
•tRNA – transfer RNA: molecules that transport
amino acids to ribosomes during protein synthesis
•rRNA – ribosomal RNA: short single stranded RNA
molecules and are components of ribosomes
•Ribosomes – site of protein synthesis
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•How is mRNA read?
–Genetic code – universal language of genetics used by
virtually all living organisms
•Works in three nucleotide units of mRNA called codons
•Each codon codes for a single amino acid
•One amino acid may be coded for by more than one codon
–There are 64 codons and only 20 amino acids – degeneracy of the
genetic code
•Start codon (AUG) codes for Met and signals starting point for translation
•Stop codons – UGA, UAA, UAG – do not code for amino acid but signal
end of translation
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2.4 RNA and Protein Synthesis
•Read the genetic code and state which amino acid has the most
codons. Explain.
• Read the genetic code and state which amino acid has the least
amount of redundancy within the genetic code.
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•Ribosomes and tRNA molecules
•Ribosomes – aggregates containing rRNA and protein that
make up subunits
–Each ribosome contains 2 subunits: large and small and associate to
form 2 grooves A (aminoacyl) and P (peptidyl) site into which tRNA
molecules bind and also E (exit) site which tRNA molecules leave the
ribosome
–tRNA – small molecules that fold into cloverleaf structure- has site for
amino acid attachment by aminoacyl tRNA synthetase enzyme
•Aminoacyl tRNA bind to A site of ribosome
•Opposite end of tRNA is 3 nucleotide sequence = anticodon
•Each amino acid binds to a different anticodon
• Anticodons form complimentary base pairs with what part of the
mRNA sequence?
•What are the 3 players of translation?
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
Stages of translation
1.Initiation – small ribosome subunit binds to 5' end of mRNA
•What part of mRNA processing allows this 5' end to be recognized by
ribosome?
–Initiation protein factors help guide small ribosome subunit to mRNA
–Small ribosome subunit moves along the mRNA until the start codon is found
–Small subunit waits for correct tRNA (initator tRNA)
• Met amino acid is attached to what anticodon?
–Now large subunit binds to complex containing small subunit; initiation factors;
mRNA; initiator tRNA
2. Elongation – tRNAs, carrying the correct amino acid, enter the ribosome, one
at a time, as the mRNA code is read after 2 tRNAs are attached to ribosome
peptidyl transferase catalyzes formation of peptide bond between 2 amino acids
translocation phase: ribosome shifts so tRNA and protein move into P site
3. Termination – ribosome encounters the stop codon
–Releasing factor proteins interact stop codon to terminate translation and
ribosomal subunits come apart and release the mRN with newly formed protein
released into the cell
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2.4 RNA and Protein Synthesis
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2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–Gene expression refers to the production of mRNA by a cell
•All cells of an organism contain the same genome.
•If they have the same genome, then why are pancreas
cells different from lung cells?
•Not all genes will be turned on at the same time – some will
be upregulated while others will be silenced
•Will a gene coding for a protein that is involved in
concentrating be turned on while you are in this lecture
and while you are sunbathing at the beach? Work in
groups to discuss the answer.
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–Gene regulation is how genes can be turned on
and off in response to different signals
–There are several levels of gene regulation
–The fastest gene regulation is energetically the
most costly. Work in groups to explain this idea.
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2.4 RNA and Protein Synthesis
•Levels of gene expression
regulation
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2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–Transcriptional regulation – controlling the amount of
mRNA transcribed from a particular gene as a way to turn
genes on or off
–Promoter is DNA sequence located upstream from the gene
•Certain sequences found in the promoter region
–TATA box and CAAT box
•RNA polymerase cannot bind to promoter region without presence of
transcription factors – DNA binding proteins
•Enhancer DNA sequences which are located very far upstream or
downstream of a gene can bind to regulatory proteins called activators
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2.4 RNA and Protein Synthesis
•How can transcription be inhibited? Work in groups to answer this
question.
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2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–Bacteria use operons to regulate gene expression
•Organization of bacterial genes
•Clusters of several related genes located together and
controlled by a single promoter
•Operator – region within promoter
–Can use operons to regulate gene expression in
response to their nutrient requirements
•lac operon
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2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–Bacteria use operons to regulate gene expression
•Organization of bacterial genes
•Clusters of several related genes located together and
controlled by a single promoter
–Can use operons to regulate gene expression in
response to their nutrient requirement
© 2013 Pearson Education, Inc.
2.4 RNA and Protein Synthesis
•Lac operon and its players:
•Laci codes for repressor protein
•3 genes coded by lac operon: lac z codes for B Gal that breaks down
lactose into glucose and galactose; lacy codes for galacto permease
which is a membrane protein that transports lactose into the cell; lacA
codes for acteyltransferase which has a protective function
•When there is lactose, it acts as an inducer by binding to the repressor
and changing its shape so it falls off the operator and allows RNA Pol to
transcribe the 3 genes
•When there is glucose the repressor stays bound and prevents
transcription
•Why is it energetically favorable to not transcribe the 3 genes
coded by the lac operon when the bacteria is exposed to glucose
and no lactose?
•Why is it necessary to turn transcribe the 3 genes coded by the lac
operon in the presence of lactose and no glucose?
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2.4 RNA and Protein Synthesis
lac operon
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2.4 RNA and Protein Synthesis
•Basics of Gene Expression Control
–RNA interference – RNA mechanism of gene silencing
–Short interfering RNA (siRNA) – 22 nucleotide double
stranded non-coding RNA bind to mRNA and regulate gene
expression by "silencing" gene expression through blocking
or interfering with translation of bound mRNA
–Micro RNA – another category of small RNA molecules that
do not code for proteins
•miRNA silence gene expression by blocking translation of mRNA or
causing degradation of mRNA
•Work in groups and describe an example of how RNA interference
can be used as a gene therapy for a particular disease.
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2.4 RNA and Protein Synthesis
RNA interference
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2.5 Mutations: Causes and Consequences
•Mutation – change in the nucleotide sequence of DNA
–Major cause of genetic diversity
–Can also be detrimental
–Can be spontaneous (due to error during DNA replication) or induced
due to environmental factors including X rays and UV
–They exert their effects on a cell by changing properties of the protein
which affects the trait
•Types of Mutations
•Work in groups to discuss examples of each type of
point mutation and how it can affect the protein structure
and function
–Point mutations
•Silent mutations
•Missense mutations
•Nonsense mutations
•Frameshift mutations
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2.5 Mutations: Causes and Consequences
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2.5 Mutations: Causes and Consequences
•Gene mutations can be inherited or acquired
–Inherited mutations are those passed on to
offspring through gametes
•Can cause birth defects or inherited diseases because the
mutation is present in the genome of all of the offspring's
cells
–Acquired mutations occur in the genome of
somatic cells
•Are not passed along to offspring
•These mutations can lead abnormalities in cell growth and
ultimately become cancerous
© 2013 Pearson Education, Inc.
2.5 Mutations: Causes and Consequences
•Mutations are a major cause of genetic
diversity
–Human genomes are approximately 99.9%
identical
•0.1% differences in DNA between individuals, or one base
out of every thousand
–Roughly 3 million differences between different individuals
•Most have no obvious effects; other mutations strongly
influence cell functions, behavior, and susceptibility to
genetic diseases
•Most genetic variations are created by SNPs (single
nucleotide polymorphism) – are bad when they occur in
exons and change protein structure so ultimately can change
protein function
© 2013 Pearson Education, Inc.
2.6 Revealing the epigenome
•Epigenome – modifications in chromatin structure
which DO NOT involve mutations in DNA sequence
•Changes in epigenome affect both DNA and
histones
–Some epigenetic modifications are inherited and others vary
from generation to generation
–Some are reversible; some are long lasting
–These changes vary between cell types in the body and in
normal and diseased tissues
–Diet and environment can influence epigenome
–Involved in patterns of gene expression during development
© 2013 Pearson Education, Inc.
2.6 Revealing the epigenome
•Please watch the interactive video:
http://learn.genetics.utah.edu/content/epigenetics/
twins/epigenetics.
•Then work in groups and discuss why identical twins
are different as they get older.
© 2013 Pearson Education, Inc.
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BIOTECHNOLOG
UNIT TWO: Raw Materials and Basic Skills
BIOTECHNOLO
GY
UNIT TWO: Raw Materials and Basic Skills