NOTE SET 9 (Exam 4 material)
Chapter 17 - RNA and Protein Synthesis
“From Gene to Protein”
Connection between genes and proteins
Synthesis and Processing of RNA
– Transcription
Synthesis of Protein
– Genetic Code
– Translation
Genetic Information
Stored in DNA
– the sequence of bases
– genes: scattered along chromosomes
Genetic info dictates synthesis of proteins
Proteins are the links between genotype (genetic makeup) and phenotype (appearance)
Connection between genes and proteins
Beadle and Tatum (and others previously)
– studied mutations in genes
– organism = Neurospora crassa (bread mold)
Auxotrophs - metabolic defects
– can grow on complete medium
– but not on minimal medium
– grow mutants on minimal medium with specific supplements
• (vitamin or amino acids)
– group based on growth characteristics
Beadle and Tatum’s Experiment
The one gene-one enzyme hypothesis
Beadle and Tatum’s Conclusions
Beadle and Tatum (and others previously)
– Altered genes = Altered enzymes (proteins)
– One gene - one enzyme hypothesis
Have altered the concept with time
– One gene - one protein.....TO:
– One gene - one polypeptide
Connection between genes and proteins
Central dogma of molecular genetics
DNA  RNA  protein
Overview of Transcription and Translation
Transcription
– Synthesis of RNA under the direction of DNA (DNA  RNA)
Translation
– Synthesis of a polypeptide by a ribosome under the direction of a mRNA (mRNA  protein)
Fig 17.3 Overview of Transcription & Translation
Txp and Txl are coupled in bacteria
Txp occurs in the nucleus in eukaryotes, while txl takes place in cytoplasm
Primary transcript (hnRNA) is modified (post-transcriptional modification) before being transported to
cytoplasm.
Overview of Transcription and Translation
Prokaryotic / Eukaryotic differences
– Transcription and translation are coupled in prokaryotes
– Not coupled in eukaryotes - transcription in nucleus, translation in cytoplasm
– Eukaryotic mRNAs are processed; prokaryotic mRNAs are not.
Transcription
Messenger RNA (mRNA) is transcribed from the template strand of a gene
RNA polymerase
– separates or “melts” the DNA strands
– links the RNA nucleotides as they base-pair along the DNA template.
– adds nucleotides only to the 3’ end of the growing polymer
– gene (template strand) is read 3’5’, creating a 5’3’ RNA molecule
How does the RNA polymerase know where to start and stop transcription?
– more DNA in genome than that occupied by genes
Beginning and ending of gene
– marked in DNA by specific sequences
Promoter
– RNA polymerase binds and initiates transcription
– “upstream” of the information contained in the gene, the transcription unit
Terminator
– signals the end of transcription
RNA Polymerases
Bacteria
– single type of RNA polymerase that synthesizes all RNA molecules.
Eukaryotes
– three RNA polymerases (I, II, and III)
– RNA Pol II is used for mRNA synthesis.
Fig 17.7 Stages of Transcription
Fig 17.8 Promoters
In eukaryotes, proteins called transcription factors recognize the promoter region, especially a TATA
box, and bind to the promoter.
After they have bound to the promoter, RNA polymerase binds to transcription factors to create a
transcription initiation complex
Assembling the Transcription Complex
The first transcription factor (TF) to bind recognizes the TATA box Then other TFs can bind
Fig 17.8 Transcription Initiation Complex
Close-up of Transcription Elongation
• Polymerase unwinds helix
•
•
•
Adds nucleotides that are complementary to bases in template strand
Helix rewinds after RNA polymerase passes
Many polymerase molecules can transcribe a single gene at the same time.
Transcription Termination
In prokaryotes
– specific sequence is terminator
– RNA pol stops right at sequence
In eukaryotes
– RNA pol continues for hundreds of nucleotides past the terminator sequence: AAUAAA
– another enzyme cuts the RNA 10 to 35 bases past the terminator sequence
RNA Processing in Eukaryotic Cells
Primary transcript (pre-mRNA or hnRNA) is modified before transport to cytoplasm
– 5’cap
– polyA tail
– RNA splicing (removal of introns)
Eukaryotic mRNA Processing
5’ Cap
– modified form of guanine nucleotide
• Linked via 5’ --> 5’ phosphodiester bond
– Helps protect mRNA from degradation
– Important for translation initiation
• aids in ribosome binding
PolyA tail at 3’ end
– An enzyme adds 50-250 adenine nucleotides (poly A polymerase)
– Functions
• Protects from degradation
• Important for translation
• Facilitates export of mRNA from nucleus
RNA Processing: Splicing
– Eukaryotic Genes
• Composed of alternating exons and introns
• Exons
• expressed regions
• end up in final mRNA
• Introns
• intervening sequences
• removed from mRNA
Fig 17.10 RNA splicing
• Accomplished by a protein/RNA complex called - spliceosome
– consists of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs)
– Each snRNP has several protein molecules and a small nuclear RNA molecule (snRNA).
• Each is about 150 nucleotides long.
Fig 17.11 Role of snRNPs
Alternative RNA Splicing
• Gives rise to two or more different polypeptides, depending on which segments are treated as exons.
– Early results of the Human Genome Project indicate that this phenomenon may be common in
humans.
Fig 17.12 Exons = Protein Domains
• Domains in proteins
• Discrete structural and functional regions
• Often encoded by distinct exons of gene
• May facilitate evolution via recombination between genes: “Exon Shuffling”
Overview of Translation
Genetic information stored as the nucleotide sequence is converted into an amino acid sequence
Read in groups of three nucleotides = Codons
THEFATCATATETHERAT
HEFATCATATETHERAT
EFATCATATETHERAT
Fig 17.4 The Triplet Code
mRNA is “read” in groups of three nucleotides,called “codons”
String of codons is an open reading frame (ORF)
AUG = txl start
UAA, UAG, or UGA = txl stop
64 possible codons - combinations of 3 bases
codons are read in a 5’  3’ direction
Each codon specifies which of the 20 amino acids will be incorporated
# of nucleotides is 3x the number of amino acids for a given coding region/protein sequence
# codons = # aa
Figuring out the Genetic Code
Marshall Nirenberg determined the first match in the early 1960s
UUU specifies phenylalanine
o created artificial mRNA (all uracil bases)
o translated by purified ribosomes in vitro
o produced a polyphenylalanine
Other researchers and more elaborate techniques decoded the remaining codons
Fig. 17.5 The Genetic Code Dictionary
Characteristics of the Genetic Code
Universal
o Essentially all organisms use the same genetic code dictionary
Degenerate
o As many as six codons may specify the same amino acid - See Ser, Leu, Arg
Unambigous
o A codon specifies only one amino acid
Summary
Genetic info encoded as sequence of non-overlapping base triplets, or codons
Each codon is translated into a specific amino acid during protein synthesis
Codons are read sequentially in a 5’3’ direction
Fig 17.13 Translation: The basic concept
tRNA transfers amino acids from the cytoplasm’s pool to a ribosome.
Each tRNA carries a specific amino acid at one end
Other end has a specific nucleotide triplet - anticodon that basepairs with codons in mRNA
Rribosome adds the growing polypeptide chain to the next amino acid carried by tRNA that is bound
to the ribosome
Fig. 17.14 The Structure of tRNA
tRNA and Wobble Hypothesis
61 codons specify amino acids
But only about 45 tRNAs!!
Anticodons of some tRNAs can recognize two or more codons
U in anticodon can base pair with either A or G in codon
I (Inosine, a purine) in anticodon can basepair with U, C, or A in codon
Some anticodons can recognize two or more codons
U in anticodon can base pair with either A or G in codon
AAU ---> UUG,UUA
I (Inosine) in anticodon can base pair with A, C, U
CCI ---> GGA,GGC,GGU
Wobble Hypothesis
Affects basepairing of 3rd base of codon only
1st and 2nd base of codon follow Watson-Crick base pairing A=U, G=C
Fig 17.15 Joining of a specific amino acid to a tRNA by aminoacyl-tRNA synthetase
Amino acyl-tRNA synthetase links amino acid to specific tRNA
– 20 different enzymes - one for each amino acids
Note: ATP hydrolyzed to AMP in “charging” tRNA with amino acid. Effectively, 2 ATP
consumed
Fig 17.16 The anatomy of a ribosome
Each ribosome has binding site for mRNA and 3 binding sites for tRNAs
• P site holds tRNA that carries growing protein
• A site carries tRNA with next amino acid to come in
• Discharged tRNAs leave ribosome at E site.
Comparison of Ribosomes
Stages of Translation
Initiation, Elongation, Termination
Fig 17.17 Initiation of translation
Start codon is AUG
Translation Elongation
Translocation
o Ribosome moves the tRNA with the attached polypeptide from the A site to the P site
or one codon)
o Requires hydrolysis of GTP
o tRNA still basepaired to mRNA, so mRNA also moves
o tRNA in P site moves to the E site and then leaves the ribosome
Fig 17.18 Translation Elongation
Fig 17.19 Termination of Translation
Stop codon reaches A site
Release Factor binds to stop codon
(3 nucleotides
Hydrolyzes bond between polypeptide and tRNA in P site
Translation complex disassembles
Fig 17.20 Polyribosomes
More than one ribosome may translate an mRNA at the same time, so many copies of a protein molecule
may be obtained from one mRNA
Ribosomes and Translation
Ribosomes
Cytosolic
Membrane-bound - rough ER
Protein Secretion
rough ER --> Golgi --> Secretory Vesicles
What determines whether a cytosolic or rER-bound ribosome will translate an mRNA?
Signals for Protein Secretion
Signal peptide at start of coding region of polypeptide targets ribosome and mRNA to rER.
Fig 17.21 Signal Mechanism for Targeting Proteins to the ER
Signal Peptides
Other types of signal peptides target proteins to other organelles:
mitochondria
chloroplasts
nuclei
Multiple Roles for RNA in Cells
mRNA
carries info from DNA to ribosome
rRNA
structural and catalytic role in ribosome
tRNA
adapter molecule in protein synthesis
1° transcript first RNA - prior to processing or splicing
snRNA
in spliceosomes - structural & catalytic
SRP RNA
component of signal recognition particle
Sno RNA
Aids in processing pre-rRNA
siRNA/miRNA
involved in regulation of gene expression
Connection between genes and proteins
Genetic information is stored in DNA
As the nucleotide sequence
Proteins are the expressed form of the genetic information
Mutations
Changes in the genetic information in DNA
Altered nucleotide sequence
May affect proteins
Mutations in DNA
Point Mutations
change in just one base pair
basepair substitution
Frameshift Mutations
Due to loss of base pair(s)
Or addition of base pair(s)
Could lead to new codons (missense)
Mutations in DNA (see Fig 17.24)
Silent Mutation
point mutation that has no effect on protein sequence (generally in 3rd base of codon)
UGU (Cys) --> UGC (Cys)
Missense Mutation
point mutation that changes the amino acid
UGU (Cys) --> UGG (Trp)
Nonsense Mutation
amino acid codon altered to a stop codon
UGU (Cys) --> UGA (Stop)
Fig 17.23 The molecular basis of sickle-cell disease
Erythrocyte Phenotypes
Other Types of Mutations
Insertions/Deletions
Additions or losses of one or more nucleotide pairs
May causes a “frameshift” in the open reading frame (ORF)
Nucleotides read in new combinations of triplets - new codons
Fig 17.25 Consequence of bp deletion, insertion, codon insertion
Chapter 25 - The History of Life on Earth
Overview: Lost Worlds
The fossil record shows macroevolutionary changes over large time scales including
The emergence of terrestrial vertebrates
The origin of photosynthesis
Long-term impacts of mass extinctions
Conditions on early Earth made the origin of life possible
Chemical and physical processes on early Earth may have produced very simple cells through a sequence
of stages:
1. Abiotic synthesis of small organic molecules
2. Joining of these small molecules into macromolecules
3. Packaging of molecules into “protobionts”
4. Origin of self-replicating molecules
Synthesis of Organic Compounds on Early Earth
Earth formed about 4.6 billion years ago, along with the rest of the solar system
Earth’s early atmosphere likely contained water vapor and chemicals released by volcanic
eruptions (nitrogen, nitrogen oxides, CO2, methane, ammonia, hydrogen, hydrogen sulfide)
A. I. Oparin and J. B. S. Haldane hypothesized that the early atmosphere was a reducing environment
Stanley Miller and Harold Urey conducted lab experiments that showed that the abiotic synthesis of
organic molecules in a reducing atmosphere is possible
The experiments of Stanley Miller (chapter 4) and others have shown that “reducing environments” rich
in amino acids, nucleic acids, lipids, and carbohydrates would have been abundant on primordial earth.
However, the evidence is not yet convincing that the early atmosphere was in fact reducing
Instead of forming in the atmosphere, the first organic compounds may have been synthesized near
submerged volcanoes and deep-sea vents
Amino acids have also been found in meteorites
Abiotic Synthesis of Macromolecules
Small organic molecules polymerize when they are concentrated on hot sand, clay, or rock
Protobionts - aggregates of abiotically produced molecules surrounded by a membrane or membrane-like
structure
Protobionts exhibit simple reproduction and metabolism and maintain an internal chemical environment
Experiments demonstrate that protobionts could have formed spontaneously from abiotically produced
organic compounds
e.g. small membrane-bounded droplets called liposomes can form when lipids or other organic molecules
are added to water
Self-Replicating RNA and the Dawn of Natural Selection
First genetic material probably RNA, not DNA
RNA molecules called ribozymes have been found to catalyze many different reactions
e.g., ribozymes can make complementary copies of short stretches of their own sequence or other short
pieces of RNA
Early protobionts with self-replicating, catalytic RNA would have been more effective at using resources
and would have increased in number through natural selection
Early genetic material might have formed an “RNA world”
First Single-Celled Organisms
Oldest known fossils are stromatolites, rock-like structures composed of many layers of bacteria and
sediment
Stromatolites date back 3.5 billion years
Prokaryotes were Earth’s sole inhabitants from 3.5 to about 2.1 billion years ago
Photosynth & the O2 Revolution
Most atmospheric O2 is of biological origin
O2 produced by oxygenic photosynthesis reacted with dissolved iron and precipitated out to form banded
iron formations
Source of O2 likely bacteria similar to modern cyanobacteria
~2.7 billion years ago, O2 began accumulating in atmosphere and rusting iron-rich terrestrial rocks
This “oxygen revolution” (2.7 to 2.2 billion years ago)
Posed a challenge for life
Provided opportunity to gain energy from light
Allowed organisms to exploit new ecosystems
The First Eukaryotes
The oldest fossils of eukaryotic cells date back 2.1 billion years
The hypothesis of endosymbiosis proposes that mitochondria and plastids (chloroplasts and related
organelles) were formerly small prokaryotes living within larger host cells
An endosymbiont is a cell that lives within a host cell
The prokaryotic ancestors of mitochondria and plastids probably gained entry to the host cell as
undigested prey or internal parasites
In the process of becoming more interdependent, the host and endosymbionts would have become a single
organism
Serial endosymbiosis supposes that mitochondria evolved before plastids through a sequence of
endosymbiotic events
Key evidence supporting an endosymbiotic origin of mitochondria and plastids:
Similarities in inner membrane structures and functions
Division is similar in these organelles and some prokaryotes
These organelles transcribe and translate their own DNA
Their ribosomes are more similar to prokaryotic than eukaryotic ribosomes
The Origin of Multicellularity
The evolution of eukaryotic cells allowed for a greater range of unicellular forms
A second wave of diversification occurred when multicellularity evolved and gave rise to algae, plants,
fungi, and animals
Earliest Multicellular Eukaryotes
Comparisons of DNA sequences date the common ancestor of multicellular euk. to 1.5 billion yrs ago
Oldest known fossils of multicellular eukaryotes are of small algae that lived ~1.2 billion yrs ago
The “snowball Earth” hypothesis suggests that periods of extreme glaciation confined life to the
equatorial region or deep-sea vents from ~750- 580 million yrs ago
The Cambrian Explosion
The Cambrian explosion refers to the sudden appearance of fossils resembling modern phyla in the
Cambrian period (535 to 525 million years ago)
The Cambrian explosion provides the first evidence of predator-prey interactions
So does that mean all life came from ONE original cell?
Not exactly, but all 3 domains of life probably had a
“last universal common ancestor”
The first organism was probably an RNA-based (rather than DNA) life form with extensive amounts of
horizontal genetic transmission (viral infection?) between cells. The evolution of a reverse transcriptase
or transposon-like activities may have converted the RNA based life form into DNA-based cell types--at
least 3 types of which survive today.