Chapter 6: Molecular information flow and protein processing Flashcards
gene
Functional unit of genetic information
genetic elements
Genes are part of genetic elements: large
molecules and/or chromosomes
Genome
all genetic elements
Informational macromolecules
nucleic acids and
proteins
Nucleotides
nucleic acid monomers
D N A and R N A are polynucleotides
Three components: pentose sugar (ribose or
deoxyribose), nitrogenous base, phosphate (PO43−)
DNA and RNA
DNA (genetic blueprint) and RNA (transcription
product)
Messenger RNA (mRNA) is translated into
protein (amino acid sequence)
Nucleoside
has pentose sugar and
nitrogenase base, no phosphate
Properties of the Double Helix
Nucleic acid backbone is a polymer of alternating phosphates and the pentose sugar deoxyribose
Phosphodiester bonds connect 3′-carbon of
one sugar to 5′-carbon of the adjacent sugar
DNA is double-stranded and held together by hydrogen bonding between bases
Two strands are antiparallel (5′-3′ and 3′ to 5′),
forming double helix
Contains two grooves, major (where proteins bind)
and minor
Primary structure (DNA)
sequence of nucleotides
that encodes genetic information
complementary base sequences
Adenine pairs with thymine
Guanine pairs with cytosine
Size and Shape of DNA
Size is expressed in number of nucleotide base pairs
1000 base pairs = 1 kilobase pair = 1 kbp
1 million base pairs = 1 megabase pair = 1Mbp
E. coli genome = 4.64 Mbp
Linear DNA length is several hundred times longer than cell, so supercoiling compacts DNA to
accommodate genome
supercoiled DNA mechanism
Supercoiled DNA components
Topoisomerases insert and remove supercoils
Negative supercoiling: twisted in opposite sense relative to right-handed double helix; found in most cells
DNA gyrase: introduces supercoils into DNA via double-strand breaks
Positive supercoiling: helps prevent DNA melting at high temperatures (e.g., some Archaea)
Central dogma
Genetic information flow can
be divided into three stages, DNA to RNA to
protein
Gene expression transfers DNA information to
RNA.
Three main RNA classes involved in protein synthesis
mRNA (messenger RNA): carry information to ribosome
tRNA (transfer RNA): convert mRNA information to amino acid sequence
rRNA (ribosomal RNA): catalytic and structural ribosome components
Synthesis of informational macromolecules steps
Eukaryotic genetic information flow
Each gene is transcribed individually into a single mRNA ( Monocistronic mRNA)
Replication and transcription occur in nucleus
RNAs must be exported outside nucleus for translation
Many different RNAs can be described from a short DNA region
Prokaryotes genetic information flow
Multiple genes may be transcribed in one m RNA (Polycistonic mRNA)
Coupled transcription and translation occur producing proteins at maximal rate
How would a virus violate the
central dogma?
Viruses go backwards
contain RNA converted into DNA by reverse transcriptase
Chromosome
main genetic element in prokaryotes
Other genetic elements include virus genomes, plasmids, organellar genomes, and transposable
elements
Most Bacteria and Archaea have single circular chromosome carrying all/most genes
Eukaryotes: two or more linear chromosomes
Kinds of genetic elements summary
Viruses and plasmid DNA content
Viruses contain either RNA or DNA genomes.
can be single- or double-stranded
can be linear or circular
Plasmids: circular or linear double-stranded DNA that replicate separately from chromosome
Cell can survive without plasmid but survives better with it
Replicates separately from chromosomes
Transposable elements
segments of DNA inserted into other DNA molecules that can move from one site to another site on the
same or a different DNA molecule (e.g., chromosomes, plasmids, viral genomes)
found in prokaryotes and eukaryotes
Some features of the Escherichia coli K-12 chromosome
About 5 Mbp in size
Almost 4300 possible protein-encoding genes make up 88 percent of the genome
Compact relative to eukaryotes, which contain extra DNA
Many genes encoding enzymes of a single biochemical pathway are clustered into operons, transcribed to form single
mRNA and regulated as a unit
Many genes for biochemical pathways are not clustered
Thus, operons appear to be exceptions instead of the rule
plasmid description
found in many Bacteria and Archaea
mostly non-essential but may influence host cell physiology (e.g., survival under certain
conditions)
nearly all double-stranded DNA, mostly circular
typically, less than 5 percent of the size of the chromosome
present in different copy number (1 or a few to 100+ copies)
R plasmids
Widespread and well-studied
Resistance plasmids; confer resistance to antibiotics or other growth inhibitors
Several antibiotic resistance genes can be encoded on one R plasmid (e.g., R100)
what do plasmids code for
In several pathogenic bacteria, virulence factors (e.g., ability to attach or produce toxins) are encoded by
plasmids
Bacteriocins (proteins that inhibit or kill closely related species or different strains of the same species) can
be encoded on plasmids
Rhizobia require plasmid-encoded functions to fix nitrogen
Metabolism (e.g., hydrocarbon degradation)
Important for conjugation (horizontal gene transfer)
Verticle transfer
From parents to offspring
Plasmids can also be transferred
vertically
Type of DNA replication
semiconservative: each of
the two resulting double helices has one new
strand and one parental (template) strand
Precursor of each nucleotide is a
deoxynucleoside 5′-triphosphate
Replication ALWAYS proceeds from the 5′
end to the 3′ end
types of DNA polymerase
DNA polymerases catalyze polymerization of deoxynucleotides
Five different DNA polymerases (DNA Pol I –V) in E. coli
DNA Pol III is the primary enzyme in DNA replication
DNA Pol I plays a lesser role in DNA replication
Other repair DNA damage
Replication Enzyme mechanism
DNA polymerases can only add nucleotides to pre-existing 3′-OH and require a primer: short
stretch of RNA
Primer made from RNA by primase
Primer eventually removed and replaced with DNA
type of replication enzymes
Helicase
Single-strand binding protein
Primase
DNA polymerase III
DNA polymerase I
DNA ligase
Topoisomerase IV
Helicase
Unwinds double helix at replication fork
Single-strand binding protein
Prevents single strands from annealing
Primase
Primes new strands of DNA
DNA polymerase III
Main polymerizing enzyme
DNA polymerase I
Excises RNA primer and fills in gaps
DNA ligase
Seals nicks in DNA
Topoisomerase IV
Unlinking of interlocked circles
Initiation of DNA Synthesis
DNA synthesis begins at a specific location on the bacterial chromosome called the origin of replication (oriC).
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The protein DnaA binds to oriC and opens up the DNA double helix.
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Enzymes called DNA helicases then bind to the DNA molecule and begin to unwind the double helix, using energy from ATP. This creates two Y-shaped replication forks, which are the sites of DNA synthesis.
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Single-strand binding proteins bind to the now-separated single strands of DNA to prevent them from coming back together.
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Topoisomerases, such as DNA gyrase, help relieve stress on the DNA molecule as it unwinds by creating breaks in the DNA and then rejoining them, introducing negative supercoils.
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The enzyme primase then synthesizes a short RNA primer, providing a free 3’-OH group for DNA polymerase to attach the first DNA nucleotide. The primase is sometimes referred to as a primosome.
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Once the primer is in place, DNA polymerase III can begin adding deoxyribonucleoside triphosphates (dNTPs) to the 3’ end of the primer. This marks the beginning of DNA synthesis.
Leading and Lagging Strands and the Replication Process
DNA replication at replication fork
occurs continuously on the leading strand 5’ to 3′ (always free 3’-OH)
discontinuously on lagging strand—no 3’-OH
Primase synthesizes multiple RNA primers
Leading and Lagging Strands and the Replication Process (after replication fork has been made)
DNA replication at replication fork
Primase replaces by DNA Pol III and DNA synthesis until it reaches the previously synthesized DNA
DNA Pol I removes the RNA primer and replaces it with DNA
DNA ligase seals the nicks in the DNA
replication rate
DNA Pol III adds 1000 bp per second
Replication of Circular DNA
known as theta structure
The Replisome
large replication complex of multiple proteins
When replication forks collide at terminus of replication (opposite origin), replisome is finished
DNA is partitioned; facilitated by FtsZ
Primosome
helicase and primase subcomplex within replisome
Why do we need proofreading?
Mistakes Mutations Misfoldedprotein
Mutation rates are 10^-8 to 10^-11errorsperbp
Proofreading ensures high fidelity
Fidelity of DNA Replication:
During DNA replication DNA Pol I and III can detect a bp mismatch through double helix distortion
Can remove the mismatched bp with 3’ - 5’ exonuclease activity
Exonuclease proofreading occurs in prokaryotes, eukaryotes, and viral DNA replication systems
Two key differences in RNA vs DNA chemistry
ribose instead of deoxyribose
uracil instead of thymine
Typically, single-stranded
Fold into secondary structure (complementary base pairing) that influences function
Transcription mechanism
Catalyzed by RNA polymerase
Phosphodiester bonds between ribonucleotides
Similar mechanism
Uses DNA as template; only one strand transcribed
no priming needed
Transcription terminators mark end of transcription
Highly regulated process allowing transcription at different frequencies depending on need
Transcription binding
RNA Polymerases and the Promoter Sequence
RNA polymerase has five different subunits
forming RNA polymerase holoenzyme
complex
Sigma not as tightly bound, easily dissociates to yield
RNA polymerase core enzyme
Core enzyme synthesizes RNA
Sigma recognizes initiation sites on DNA called promoters to begin transcription
Transcription occurs in opposite directions on DNA strands
sigma factors
Promoters are specific DNA sequences
recognized by σ70
sequences vary but two highly conserved regions: Pribnow box (−10 region, TATAAT) and TTGACA (−35)
Strong promoters: promoters conforming most closely to consensus sequences more effective in binding RNA
polymerase
Alternative sigma factors recognize different consensus sequences
Transcriptional units
Transcriptional units: DNA segments transcribed into 1 RNA molecule bounded by initiation and
termination sites
can result from 1 or 2+ (co-transcribed) genes
Most genes encode proteins, but some encode untranslated RNAs (e.g., rRNA, tRNA)
e.g., three types of rRNA: 16S, 23S, and 5S + one tRNA
Operon transcription
Operons are transcribed into a single mRNA called a polycistronic mRNA containing multiple open reading frames, ( sequences that encode amino acids to make a polypeptide)
Termination of Transcription
Termination governed by specific DNA sequences
Example: GC-rich sequence containing an inverted repeat and a central nonrepeating segment followed by
several adenines
RNA forms stem-loop by intra-strand base pairing, RNA polymerase pauses, DNA and RNA dissociate
Rho-dependent termination: Rho protein recognizes specific DNA sequences (Rho-dependent
termination site) and releases RNA polymerase from DNA
No. of DNA pol of diffferent domains
Archaea contain one RNA polymerase
Resembles eukaryotic polymerase
Eukaryotes have 3 DNA polymerases
binding sites for transcription for Archae
Most important recognition sequence is 6–8 base pair “TATA” box
18–27 nucleotides upstream of transcriptional start site
recognized by TATA-binding protein (TBP) transcription factor
B recognition element (BRE), upstream of TATA box, recognized by archaeal transcription factor B (TFB)
Binding of TBP to TATA and TFB to BRE enables RNA polymerase binding and
transcription initiation
Less known about termination in Archaea
Some archaeal genes have inverted repeats followed by AT-rich sequence similar to bacterial terminators
One suspected terminator lacks inverted repeats but has repeated thymine runs
Eta transcriptional termination protein found in Euryarchaeota
Binds upstream of transcription bubble
Collides with archaeal RNA polymerase and binds it off DNA
coding and noncoding regions
exons: coding sequences
introns: intervening noncoding sequences
found in tRNA and rRNA genes of Archaea
RNA processing of primary transcript (original transcribed RNA) required to form mature RNAs for translation
splicing in Eu and Arc
Eukaryotes: splicing occurs in nucleus via the
spliceosome (RNA + protein)
Archaea: introns rare in protein encoding-genes but
need to be removed from tRNA- and rRNA-encoding
transcripts by special ribonuclease
splicing definition
removing introns and joining exons
splicing mechanisms
steps in eu processing before splicing
capping: addition of methylated guanine to 5′ end of mRNA in reverse orienation, needed to initiate translation
polyadenylation: trimming 3′ end addition of 100–200 adenylate residues (poly(A) tail) to stabilize mRNA; must be
removed before mRNA can be degraded
AA composition
Proteins are polymers of amino acids: organic
compounds containing both an amino (-NH2) and
carboxylic acid (-COOH) linked to and α-carbon
Amino acids are linked by peptide bonds through
carboxyl carbon of one amino acid and amino nitrogen
of a second
amino acid side chain
Side chain abbreviated R is bonded to α-carbon
Chemical properties of amino acids are related to their side chain (e.g., acidic, basic, nonpolar)
Considerable variability
Chemically related side chains show similar properties and are grouped into “families”
different structures of proteins
Linear array of amino acids in a polypeptide called its primary structure
Once formed, a polypeptide folds to form a more stable structure
Secondary structure: from hydrogen bonding (alpha-helix or beta sheet)
Tertiary structure: three-dimensional shape of polypeptide from hydrophobic and other interactions
Quaternary structure: number and types of polypeptides (subunits) that make a protein
Denaturation
loss of structure and biological properties
tRNA structure and synthesis
Transfer RNAs (tRNAs) carry amino acids to translation machinery
Each contains an anticodon: three bases that recognize codon (three nucleic acids encoding an
amino acid)
tRNA and correct amino acid brought together by aminoacyl-tRNA synthetases
tRNA of different species
Prokaryotes have ~60 different tRNAs
Human cells have 100–110 different tRNAs
Single-stranded
Extensive secondary structure
Contain bases modified post-transcription (e.g., pseudouridine, inosine, others
General structure of Transfer RNA
Extensive double-stranded regions formed by internal base pairing
Cloverleaf shape
3′ end always has CCA added by CCA-adding enzyme instead of being encoded
tRNA synthesis mechanism
Recognition of tRNA by aminoacyl-tRNA synthetase is critical for translation fidelity
Requires specific contacts
Amino acid activated by ATP to form aminoacyl-AMP
Amino acyl-AMP is attached to CCA stem of tRNA resulting in charged tRNA (aminoacyl-tRNA)
Aminoacyl-tRNA leaves synthetase and will be bound ribosome
codon
A triplet of nucleic acid bases (codon) encodes a specific amino acid
64 possible codons
specific codons for starting and stopping translation
Degenerate code
Multiple codons encode a single amino acid (64 codons versus 22 natural amino acids);
lacks one-to-one correspondence
Codon recognition
Codon recognition occurs by specific base pairing with complementary anticodon sequence on tRNA
Some tRNAs recognize more than one codon
Wobble: irregular base pairing allowed at third position of tRNA
Codon bias
Multiple codons for the same amino acid are not used equally
varies between organisms
correlated with tRNA concentration
Start and Stop Codons and Reading Frames
Start codon: Translation begins with AUG, encodes N-formylmethionine in Bacteria and methionine
in Archaea and Eukarya
Reading frame: Triplet code requires translation to begin at the correct nucleotide.
Shine–Dalgarno sequence, or the ribosome-binding site (RBS), ensures proper reading frame in
Bacteria
Stop (nonsense) codons: terminate translation (UAA, UAG, and UGA)
Sometimes unusual amino acids selenocysteine and pyrrolysine can be encoded by stop/nonsense codons
Open reading frame
AUG followed by a number of codons and a stop codon
steps for protein synthesis
Three major steps: initiation, elongation, termination
Uses mRNA, tRNA, ribosomes
Requires multiple proteins
Needs guanosine triphosphate (GTP) for energy
Ribisomes
Ribosomes: large complexes of proteins and RN
A where proteins are biosynthesized
thousands of ribosomes per cell
composed of two subunits (30S and 50S in
prokaryotes) to form 70S ribosome
S = Svedberg units
30S contains 16S rRNA + 21 proteins
50S contains 5S + 23S rRNA + 31 proteins
Translation factors (proteins) also essentia
Initiation Complex (translation)
Initiation begins with free 30S ribosomal subunit
Initiation complex (30S subunit, m RNA, formylmethionine t RNA, initiation factors (I F1, I F2, I F3) forms
50S ribosomal subunit added = 70S ribosome
Ribosome binding site: 3–9 nucleotides toward 5ʹ end of m RNA, complementary to sequences on 3ʹ end of
16S rRNA
Base pairing holds ribosome-mRNA complex in frame
initiation summary
two ribosomal subunits + formylmethionine tRNA + initiation factors assemble with mRNA
begins at an AUG start codon
elongation summary
amino acids brought to the ribosome and added to the growing polypeptide
occurs in the A (acceptor) and P (peptide) sites of ribosome
Elongation mechanism
tRNAs interact at A (acceptor) and P (peptide) sites on 50S
A site: incoming charged tRNA first attaches; loading assisted by elongation factor EF-Tu
P site: growing polypeptide chain is attached to prior tRNA
Growing polypeptide moves to tRNA at the A site as new peptide bond is formed
After elongation, tRNA holding peptide transferred to P site
Ribosome advances 3 nucleotides (1 codon) along m RNA per translocation
Amino acid-free tRNA pushed to E (exit site) and released from ribosome
Polysomes
a complex formed by multiple ribosomes simultaneously translating a single mRNA
Termination occurs at stop codon
Termination of translation
Termination occurs at stop codon
Release factors (RFs): recognize stop codon and cleave polypeptide from tRNA
Ribosomal subunits dissociate
Subunits free to form new initiation complex and repeat process
Role of Ribosomal RNA in Protein Synthesis
16S rRNA facilitates initiation via base pairing, holds mRNA in position on either side of A and P sites
Also involved in ribosome subunit association and positioning tRNAs on ribosome
Catalyzes peptide bond formation
peptidyl transferase reaction catalyzed by 23S rRNA
Involved in translocation
Interacts with elongation factors
Freeing trapped Ribosomes and Trans-Translation
ribosomes trapped if no stop codon at end of mRNA
Release factor cannot bind, ribosome cannot be released = “trapped”
Trans-translation: produces small RNA (tmRNA) that frees stalled ribosomes
Mimics both tRNA (carries alanine) and mRNA (contains stretch that can be translated)
Binds next to defective mRNA, allowing protein synthesis to proceed
Also encodes degradation signal
