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Foundational genetic > Genes, Genomes and Gene Expression > Flashcards

Genes, Genomes and Gene Expression Flashcards

1
Q

The evolving concept of the gene

A

Different representations of a gene which are all inter-related
•The gene as a unit of inheritance that determines a trait/phenotype
•The gene as physical entity carried on a chromosome
•The gene as a stretch of DNA sequence that is expressed as a gene
product
•The gene as one component of an individual’s genetic make-up
(genome)

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2
Q

genome definition

A

the genetic material that provides a set of hereditary instructions to construct and maintain an organism

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3
Q

Primary requirements of a genome

A

A genome is arranged into chromosomes, which encode sequences for:
§synthesis of RNA and cellular proteins
§DNA replication
§segregation of chromosomes during cell division
§compaction of chromosomes

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4
Q

Variation in the size of genomes

A

C-value: amount of DNA (pg)/haploid cell or number of kilobases (kb)/haploid cell
genome size of an organism does scale with the complexity of an organism
But….correlaNon is not precise especially in eukaryotes –C-value paradox
Prokaryotes <10-Mb
Simple eukaryotes <50-Mb
Complex eukaryotes >50-Mb

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5
Q

Parasitic genomes

A

Parasites generally have small genomes
Viruses are obligate parasites and use the host’s molecular machinery to reproduce
Viral genomes typically do not encode genes for:
energy production
genome replication
protein synthesis
cell division
Viral genomes typical encode genes for:
coat polypeptides
different life history traits – lysogeny vs lytic pathways

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6
Q

Bacteriophage genomes as an example

A 
Enormous range in genome structure:
Nucleic acids: RNA or DNA
Strandedness: single or double 
Shape: linear or circular molecules, sometimes segmented (multiple pieces)
Size: 2-kb to 2.5-Mb
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7
Q

Organization of genes within the viral genome

A

Many viruses have genomes in which genes overlap
-First DNA-based genome to be sequenced -Fred Sanger (1977) fX174 viru
-Two different transcripts from the same genomic region
Transcripts are translated from different start positions

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8
Q

Location of DNA in bacteria

A 
Genome is composed of chromosomal DNA located in a membrane-less region of the  cytoplasm –nucleoid
Extrachromosomal DNA (plasmids) located in the cytoplasm
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9
Q

Features of bacterial chromosomes

A

Chromosomes are usually circular, but some are linear
Number of chromosomes may vary between species –unipartite vs multipartite
Genetically haploid –1 copy of each chromosome
Chromosome number does not correlated with gene number

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10
Q

Structure (shape) of bacterial chromosomes

A

Chromosome needs to be condensed approx. 1,000-fold to fit into the cell
looping (10-fold compaction), supercoiling (100-fold compaction)
looping:
-Base of loops anchored to a protein core (NAPs)
-60 bp wound around a protein tetramer
Supercoiling:
•Reduced/additional turns introduced into DNA helix
•Torsional stress reduced by DNA winding around itself (supercoils)

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11
Q

Gene organization in bacterial genome

A

§Highdensity of genes
§Genes are frequently arranged into operons
§Non-coding sequences are typically associated with gene regulation
§Low level of repetitive DNA (<1% of the genome)

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12
Q

Origin of replication

A

Chromosomal DNA needs to be replicated prior to cell division
Replication is initiated at the origin of replication (oriC)
oriCis also required for chromosomal segregation at cell division
DNA replication is bidirectionaland occurs at two replication forks that form a bubble
Eukaryotic chromosomes must have sequences for:
1. DNA replication –origin (ori)
2. Replication for the ends of linear chromosomes oriC occur every 100-kb in eukaryotic chromosomes

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13
Q

Plasmids

A

Autonomously replicating extrachromosomal DNA molecules found in a wide range of bacteria
Features:
Non-essential
Encode a range of functions
Generally circular double-stranded DNA molecules 2-200kb
Low (1-2) to high (>500) copies per cell
Transferred from cell to cell – cross species boundaries

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14
Q

Traits encoded by plasmids

A

Bacteriocins
Plasmid encodes toxins that kill other bacteria (bacteriocin) but provide resistance in the host bacterium
Pathogenicity
Plasmid encodes functions required for infection – cholera toxin, food-poisoning, crown gall disease
Catabolism
Plasmid encodes enzymes for degradation of organic molecules – camphor, toluene
F – Fertility
Plasmid encodes genes required for conjugation, formation of Hfr by integration into chromosome
R – Resistance
Plasmid encodes genes for conjugation and resistance to antibiotics, heavy metals, antiseptics

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15
Q

the troublesome R plasmid

A
  • Antibiotic resistant genes are located within transposons on R plasmids
  • R plasmids are transmitted between bacteria via conjugation (horizontal gene transfer)
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16
Q

Location of DNA in a eukaryotic cell

A

Eukaryote genomes are composed of nuclear and organelle DNA

Phenomenon of uniparental/cytoplasmic inheritance suggests that some genetic material resides outside the nucleus

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17
Q

Organelle genomes

A

Mitochondrial (Mt) and chloroplast (Cp) genomes are found in nucleoids
Multiple copies of chromosomes within each nucleoid
Multiple nucleoids per organelle
All Cp genomes and most Mt genomes are circular, double-stranded DNA and supercoiled
Chromosomes do not encode all the proteins found in the organelle
Cp genomes similar in size and gene content (120 genes, land plants)
Mt genomes display greater variation in size but similar gene content (37 genes, metazoans)

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18
Q

Nuclear genomes

A

Chromosomes contained within a nuclear membrane
Typically multipartite –multiple linear chromosomes
Chromosomes may be present in one, two or more copies (haploid, diploid, polyploid)
Chromosome number is NOT related to organism complexity or genome size

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19
Q

Chromatin

A

Nuclear DNA extensively bound to proteins - chromatin
Nucleosomes(DNA wound around a histone) associate with each other to form a more compact structure – 30-nm fibre
The 30-nm fibre is anchored into radial loops through attachment to proteins – nuclear matrix
Higher-order compaction of radial loops to form the metaphase chromosome in a dividing cell

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20
Q

Chromatin structure is dynamic

A

-Euchromatin –regions where chromatin is less condensed Gene-rich, transcriptional active, unique DNA sequences, histone acetylation
-Modification of histone tails to make them less charged
Heterochromatin –regions where chromatin is more condensed Constitutive - common to all cells – gene poor, transcriptional inactive, repetitive DNA sequences
Facultative – variable between cells – mechanism of gene regulation
Histone tail unmodified, highly charged
cHROMATIN STRUCTURE ARE NOT UNIFORM

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21
Q

Gene organisation in eukaryote vs prokaryote

A

§Significant increase in gene size (presence of introns)
§Significant increase in the amount of DNA between between genes (intergenic region)
§Amount of intergenic DNA in the genome varies between eukaryotes –explanation for the C-value paradox
(see later lecture)

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22
Q

Telomeres

A

Specialised structures at the ends of chromosomes
Contain multiple G-rich repeats of short DNA sequence e.g. TTAGGG
Prevent loss of genomic information after each round of DNA replication (see lecture on DNA replication)
Hide the chromosome ends from DNA-repair machinery

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23
Q

Centromeres

A

Condensed chromosomes have to be separated during cell division which involves spindle microtubule attachment to a specific region of the chromosome called the centromere via a kinetochore
Specialised nucleosome/s at the centromere composed of centromere
protein A (CENP-A)
Centromere sequences –typically located at a single site on a chromosome

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24
Q

DNA replication – a recap of first year knowledge unwinding

A

DNA replication requires localized unwinding and separation of the DNA strands to produce the single-stranded DNA template – replication fork (a region where the DNA molecule is being unwound)

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25
Q

Key steps in DNA replication – Strand separation

A
  • Helicase separates the two strands of DNA at the replication fork
  • Binds to lagging strand and breaks hydrogen bonds between the bases
  • Single-stranded (SSb)binding proteins prevent the parental strands from annealing
  • Helicase travels in a 5’ to 3’ direction
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26
Q

Priming for DNA synthesis

A

A 10-12bp single-stranded RNA primer is required to initiate DNA synthesis
Primase synthesizes primers for both the leading and lagging strand
Primase recruited by the helicase – ‘primosome’

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27
Q

DNA synthesis with DNA pol

A

DNA replication requires the action of two DNA polymerases III enzymes
DNA pol III has 3’ to 5’ proof-reading activity – removal of mismatched nucleotide
RNA primers are required continuously for DNA synthesis of the lagging strand
DNA polymerase extends from multiple RNA primers to generate the Okazaki fragments

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28
Q

Replication in the lagging strand (fill up the gap)

A

DNA polymerase I removes the RNA primer through 5’-3’ exonuclease activity
DNA pol I fills gap with DNA using the upstream Okazaki fragment as primer
Nicks in the newly synthesized lagging strand are repaired through the action of DNA ligase

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29
Q

The replisome

A

Proteins associated with the replication fork form a molecular machine – called a replisome
DNA pol III adds 10nt before falling off the template
bclamp + DNA pol III adds 50,000nt before falling off template

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30
Q

Initiation of DNA replication in bacteria

A

Formation of a replication fork at the oriC begins with strand separation
•DnaA protein complex recruited to oriC– induces localize melting and strand separation within the AT-rich region
•Helicases recruited to the unwound region and rapidly denatures the DNA using ATP
•Replisome recruited
Single-stranded binding (SSBs) proteins protect the ssDNA from nucleases

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31
Q

Replication bubbles

A

Two replisomes are recruited to oriC

DNA replication proceeds in two directions

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32
Q

Accessory protein – DNA gyrase

A

DNA stand separation induces torsional stress ahead of the replication fork - supercoiling
DNA gyrase (topoisomerase II) removes supercoiling
-It sever the DNA strand, removing the torsinal stress, unwind the dna and reseal it

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33
Q

Segregation of the bacterial chromosome during cell division

A

ParB proteins bind to parental oriC and newly replicated oriC mParB associates with ParA
ParA is associated with the pole
ParB dragged to the poles along to a ParA concentration mgradient
Chromosomes are separated prior to cell division

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34
Q

DNA replication in eukaryotes

A

Principle exactly the same as prokaryotic DNA replication with some added complications:

  1. Multiple oriper chromosome – need to coordinate their activity within a chromosome and across multiple chromosomes
  2. End replication problem associated with linear chromosomes

Primer missing at the end of the lagging strand
Terminal gap cannot be filled
Internal gap
Chromosome will get progressively shorter over time

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35
Q

The eukaryotic cell cycle

A

Mitosis -> G1 (Cell growth and preparation for cell cycle entry) -> S ( DNA REPLICATION)-> G2(Cell prepares for
the M phase) ->M

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36
Q

Initiation of DNA replication in eukaryote

A

Late M:
Origin Recognition Complex (ORC) binds to origin
Start of G1:
Cdc6 recruited
Helicase and Cdt1 recruited – formation of a prereplicative complex
Start of S phase:
Cdc6, Cdt1, ORC released
Cdc6/Cdt1 destroyed
Localized unwinding of DNA at the origin
DNA pol recruited (blue), replisome formed

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37
Q

Linking DNA replication with cell cycle control

A

DNA synthesis is regulated by the availability of Cdc6 and Cdt1
Cdc6 phosphorylated by Cyclin E-Cdk2 (only found in G1)
Cdc6/Cdt1 destroyed

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38
Q

Solving the end replication problem - telomerase

A

Telomerase – an RNA protein complex
Telomerase carries a short RNA molecule – complementary to the 3’ overhang
(TERC – telomerase RNA component)
Telomerase adds DNA to the 3’ overhang using short RNA as a template and the ssDNA to initiate synthesis (TERT – telomerase reverse transcriptase)
Adds more DNA to the 3’ overhang – extended 3’ end generated
Telomerase moves (translocates) along the 3’ overhang in a 3’ to 5’ direction

39
Q

DNA synthesis at the telomere

A

Extended 3’ overhang acts as a template for conventional DNA replication
Primase adds an RNA primer
DNA polymerase synthesizes DNA to fill the gap
Primer is removed and DNA ligase seals the gap

40
Q

Telomere cap

A

Single-stranded 3’ overhang is sensitive to the DNA repair pathway – needs to be protected
Single-stranded G-rich end displaces a DNA strand in a double stranded region to form a three stranded displacement (D) loop

41
Q

Chromosomal segregation during cell division (centromer action)

A

Centromere binds specialised nucleosome/s composed of centromere protein A (CENP-A)
Recruits kinetochore that allows for the attachment of the spindle machinery
Sister chromatids are pulled apart

42
Q

Stages of mitosis

A

Prophase:
Chromosomes condense.
Sister chromatids become visible and joined at the centromere
Chromosomes do NOT pair up - no recombination
Metaphase:
Spindles fibres become prominent - Chromosomes move to equatorial plane
Centromere attaches to spindle fibres from each pole
Anaphase:
Centromeres of sister chromatids ,separate
Sister chromatids move to the cell poles
Telophase:
Nuclear membrane reforms around chromosomes
Chromosomes uncoil
Formation of new cell membrane
Spindles disperse

43
Q

Classes of RNA

A

Genomes encode two main classes of RNA
Coding: messenger RNA (mRNA) rna pol1
Non-coding:
Functional – ribosomal RNA (rRNA), tRNA (transfer RNA), small nuclear
RNA (snRNA) + other classes of small RNA rna po;l 3
Regulatory – microRNA (miRNA), short-interfering RNA (siRNA), piwi-
interacting RNA (piRNA), long non-coding RNA (lncRNA) rna pol 2
Bacteria: All genes transcribed by a single RNA polymerase
Eukaryotes: Three RNA polymerases for specific classes of gene

44
Q

Organization of a bacterial protein-coding gene

A

Typical structure of protein-coding gene in bacteria:
Specific sequences define the beginning/end of the gene
Promotor where transcription star
Terminator where transcription end
Regulatory sequences upstream, downstream or in the gene determine timing and level of transcription – cis(nearby) regulatory elements bound by trans-acting(away) factors
mRNAs include protein-coding region as well as non-protein coding regions (5’ and 3’ untranslated regions)

45
Q

RNA polymerase – how it works in prokaryote strand usage and direction

A

RNA pol utilizes one of the two strands of DNA (template/non-coding strand)
RNA is ALWAYS synthesized in a 5’-to-3’ direction
Template strand is in the 3’-to-5’ orientation
RNA pol generates covalent linkages between adjacent nucleotides
RNA pol synthesis does NOT require a primer to initiate synthesis

46
Q

Anatomy of the promoter region in prokaryote

A

Promoter: Lies upstream of the transcription initiation point (+1) and coding sequences
Region that recruits RNA polymerase
Site where double-stranded DNA is separated to produce a single-stranded template for RNA polymerase
AT rich

47
Q

Promoter elements – defined by consensus

A

Promoters have two regions with conserved (highly similar) sequences
-35 box and -10 box (defined by a consensus sequence)
-35 5’ TTGACAT 3’ -10 5’ TATAAT 3’ (Prribnow box)
RNA polymerase binds directly to the DNA at these sites

48
Q

Promoter elements – defined by mutation

A

Important elements in the genome can be defined by mutagenesis – experiments on the lac operon
Look for mutants that were unable to grow on media containing lactose
Mutations reside in protein coding genes (lacZ, lacY) but also in the promoter region

49
Q

Bacterial RNA pol structure and role of S factor

A

Bacterial RNA polymerase has:
Core enzyme has 5 subunits: 2x Asubunits (formation of the complex and interaction with other regulatory protein) , 1x b(catalyse dna polymerase), b’(binding with the DNA), wsub-units (formation of the holoenzyme and gene expression)
Holoenzyme includes the sfactor which binds to -35 and -10 regions in the promoter
Sfactor contains a helix-turn-helix motif – binds to DNA
Sfactor promotes strand separation at the -10 site
Open complex formed
RNA pol initiates mRNA synthesis at the +1 site
Sfactor disassociates from the RNA polymerase complex following synthesis of a short strand of RNA
sfactor can reassociated with manother RNA polymerase core enzyme

50
Q

Steps involved in transcription – initiation in prokaryote

A

sigma factor positions the RNA polymerase for transcription initiation
Holoenzyme recognizes the promoter sequence – closed complex formed

51
Q

Elongation in prokaryote transcription

A

DNA ahead of the RNA polymerase is unwound and rewound behind the RNA polymerase – transcription bubble
Last 8-9 nucleotides added to the transcript forms a RNA-DNA hybrid
RNA pol has 3’-to-5’ proof-reading activity – error rate is 10-4
Within the bubble, if a free nucleoside triphosphate is a complementary match for the exposed base of the DNA template – RNA polymerase adds it to the RNA chain
AT/GC rule applies
Uracil instead of thymine (cheaper to made but T easier to spot for mutation)

52
Q

Mechanisms of transcription termination in prokaryote

A

TWO major mechanisms:
Factor-independent termination (Intrinsic/Rho-independent)
Rho-dependent termination

53
Q

Factor-independent termination mechanism

A

•In the template:
stretch GC-rich sequences followed by 7-8 adenines (A)
•In the RNA transcript
complementary base pair forming between GC-rich sequences formation of a stable stem-loop structure – hairpin string of uracils
RNA polymerase pauses after synthesis of the uracils (weak dna-rna link)
RNA pol backtracks to the weak RNA-DNA hybrid – encounters the hairpin
Triggers release of RNA and RNA polymerase from DNA template

54
Q

Rho-dependent termination mechanism

A

0-60bp region - C-rich + Rut (Rho utilisation) at the end of the template
Sequences causing m,RNA pol to pause
Rho (r) factor:
Homo-hexamer with helicase activity
Binds to Rut (Rho utilisation) sequences present in the mRNA
Moves towards the 3’ end of the RNA where it unwinds the RNA-DNA hybrid

55
Q

Mechanisms to ensure high levels of transcription in prokaryote

A

Multiple copies of the same gene e.g. rRNA genes
Highly active promoters – determined by regulatory sequences adjacent to the promoter
Multiple promoter on a gene

56
Q

Differences between bacteria and eukaryotes transcription (structure and location and processing)

A
  • DNA template is extensively bound by chromatin – mechanism to remove chromatin
  • Transcription and translation occur in separate cellular compartments in eukaryotes
  • RNA is synthesized in the nucleus but translated in the cytoplasm – export mechanism
  • Precursor mRNA (pre-mRNA) extensively modified in eukaryotes - RNA processing (occur co-transcriptionally
57
Q

Eukaryotic RNA pols are different to the bacterial RNA pol

A
  • There are three different RNA pols – work with distinct factors
  • RNA pols require general transcription factors (GTFs) for their recruitment to promoters
  • Distinct GTFs associate with RNA pol I, II, III promoters
  • GTFs recruit specific RNA pols to promoters
58
Q

Roles of different rna pol

A

RNA polymerase I - all rna except 5s
RNA polymerase II- protein coding rna, small nuclear rna, non-coding regulatory rna -mRNA, snRNA, snoRNAs, miRNA, long non-coding RNA
RNA polymerase III- small functioning rna- tRNA, snRNA, 5S rRNA

59
Q

Organization of a eukaryotic RNA pol II promoter

A

Core promoter is required for transcription
Alignment of eukaryotic promoters reveals the presence of conserved sequences ~30 bp upstream of transcriptional start site (+1) – TATA box (TATAAAA)
Core promoter – produces low level of transcription – basal transcription

60
Q

Organization of a eukaryotic RNA pol II promoter: Regulatory elements

A

affect the ability of RNA pol II to initiate transcription (see later)
Two category of element:
Activating sequences – enhancers
Repressing sequences – silencers Cis regulatory elements
Common position for regulatory elements is in the -50 to -100 location
Recognized by transcription factors – determine the spatial/temporal pattern of gene expression

61
Q

Requirement for Transcription initiation in eukaryote

A

Two classes of protein involved in basal transcription of genes in eukaryotes
RNA polymerase and general transcription factors (GTFs)
RNA pol II promoters require five GTFs – bind to sequences in the core promoter and/or to each other
Transcription initiation involves sequential recruitment of GTFs and RNA polymerase II

62
Q

Steps in transcriptional initiation in eukaryotes

A

Step 1 - TATA-binding protein (TBP) associated with Transcription Factor of RNA pol II D (TFIID) binds to the TATA box
Step 2 - TFIID recruits TFIIB, TFIIB recruits RNA polymerase II (RNA pol II) and another GTF
Step 3 - Two more GTFs recruited to RNA pol II Formation of the preinitiation complex is completed
Step 4 - Carboxy terminal domain (CTD) of RNA pol II is modified by one of the GTFs - release of RNA pol II from TFIIB
Most GTFs disassociate from the preinitiation complex
Step 5 – RNA pol II moves to transcriptional start site, 25bp downstream of TATA box
Remaining GTF promotes strand separation – open complex formed

Key differences to bacterial RNA pol:
RNA pol II recruited to core promoter via protein-protein interactions
RNA pol II is not associated with a s factor

63
Q

Transcription elongation regulated by cis-elements in eukaryote

A

Mediator enables TFs bound to cis-elements to influence RNA pol II activity

64
Q

Eukaryotic protein-coding genes have introns

A
  • Bacterial have a continuous open reading frame- their mRNA does not have intron
  • Eukaryote have a discontinuous reading frame which lead to the precursor rna having many non coding region
65
Q

Processing of the pre-mRNA

A

Modifications to eukaryotic mRNA requires processing steps that occur as the pre-mRNA is
synthesized in the nucleus – co-transcriptional modification
1. Capping – addition of the 5’ cap
2. Splicing – removal of introns
3. Polyadenylation – addition of a poly(A) tail
Chemical modification of RNA pol II coordinates cotranscriptional processing by creating a CTD = C-terminal domain

66
Q

Attachment of the 5’ cap

A

A methylated guanine nucleotide (m7G) present at the 5’ end of mRNA
Three step process occurs shortly after pre-mRNA emerges from RNA pol II:
1. removal a 5’ phosphate from the 5’ end of the pre-mRNA ( By TP)
2. attachment of a GMP to 5’ end of pre-mRNA (By GT)
3. attachment of a methyl group to guanine base (By MT)
5’ cap promotes exit from the nucleus and mRNA stability
5’ cap is required for translation

67
Q

Termination and polyadenylation

A

§ Conserved sequences in pre-mRNA recognized by a cleavage and polyadenylation (CP) complex (step 1) GU/U
§ CP complex contains an endonuclease - cleaves the pre-mRNA (step 2)
§ 150-200 adenine nucleotides are added to the cut end of the mRNA (step 3) by poly(A) polymerase
§ Transcription terminates 0.5-2kb downstream of the poly-A signal, cleaved 3’ end of transcript is degraded (step 4)

68
Q

RNA splicing

A

Removal of introns from the pre-mRNA and exons joined together – RNA splicing
Resulting transcript has a continuous open reading frame

69
Q

Exon-intron boundary

A 
Conserved sequences are present at the 5’ and 3’ exon-intron boundary and internally
Derive a consensus sequence – GU-AG rule
5' area have GU or GT in the DNA
3' area have AG 
Brach point have A
These are almost alway conserved
70
Q

Small nuclear RNAs and spliceosome

A

Small nuclear RNAs (snRNAs) associated with proteins - small nuclear ribonucleoproteins
snRNPs subunits are associated with different snRNAs: U1, U2, U4, U5, U6
snRNPs and other proteins form a large spliceosome complex
snRNA U1/U2 are complementary to the consensus sequences in present in pre-mRNA

71
Q

Spliceosome assembly

A

U4, U5 and U6 snRNPs sequentially join U1 and U2 snRNPs to form a spliceosome
Spliceosome formation causes intron to loop out
Adjoining exons are brought into close proximity

72
Q

splicing steps

A

U1 and U4 snRNPs released from spliceosome
TWO consecutive splicing steps – transesterification reactions
1st splicing step involves:
5’ splice site cut
5’ end of intron is COVALENTLY LINKED to A at branch site
2nd splicing step involves:
3’ splice site cut
Exon 1 covalently attached to exon 2
Intron lariat released along with snRNPs

73
Q

Types of alternative splicing

A

There is more than one method of splicing:
Exon skipping
Alternative 3’ splice sites
Alternative 5’ splice sites
Mutually exclusive exons
Many eukaryotic genes can be spliced in more than one way (alternative splicing)
Genes can encode more than one protein product – a mechanism to create
protein diversity

74
Q

Alternative splicing can produce distinct protein isoforms

A

FGFR2 pre-mRNA can be slice into two different mRNA. which cause slight variation in the end product of the protein
FGFR2 receptor isoforms bind to different ligands
FGFR2 1st isofrom regconise FGF10 FGF7
FGFR2 2nd isoform regconise FGF2 FGF8 FGF4 FGF9 FGF6

75
Q

2 process of translation

A

Translation involves two processes:
Converting a nucleotide code into a chain of amino acids – decoding function
Catalysing the formation of linkages between the amino acids to form a polypeptide – protein synthesis function

76
Q

the genetic code of amino acid

A

There are 64 possible codons with a triplet code but only 20 amino acids
Could be that:
1. 20 codons specify 20 AAs (sense) and the remaining 44 codons do not specify AAs (non-sense)
2. More than one codon specifies an AA – degeneracy
3 non-sense codons (termination or stop) UAG UGA UAA
Codons that specify the same AA are termed synonymous codons

77
Q

Codon bias

A

Degenerate sense codons are not all used equally – there is a bias that is species specific
Why is there a bias?
•Abundance of tRNA varies
•Optimize protein synthesis by selecting codons recognized by the most abundant tRNAs

78
Q

Crick’s adaptor hypothesis:

A
  1. Adaptors might contain nucleotides
  2. Adaptors could utilise base-pairing to associated with the mRNA in the same ways as nucleotides in DNA
  3. A separate enzyme is involved in joining an AA to each adaptor
79
Q

Transfer RNAs

A

tRNAs are single stranded – slightly different sequences with 3 loop, T loop to the left, anticodon loop and D loop in the right
Middle loop has three nucleotides that are antisense to a codon – anticodon
AA attached to 3’ end of tRNA
Extensive secondary structure arises through internal base-pairing – 3D L-shape

80
Q

Aminoacyl-tRNA synthetase

A

Aminoacyl-tRNA synthetases add AAs to tRNAs
Linked via the free 3’ end of tRNA and CO2-group of the AA
tRNAs with AA are called: charged
Have a specialise binding site for a AA and the tRNA

81
Q

How many aminoacyl-tRNA

synthetases are there in a cell?

A

20 synthetases….but there are more than 20 codons
Some synthetase charge more than one tRNA
Degeneracy partly involves distinct tRNAs being charged
with the SAME amino acid…………but also…….

82
Q

Some tRNAs recognize more than one codon

A

Many charged tRNAs base pair with one complementary codon
Some charged tRNAs base pair with several alternative codons
Loose base-pairing (wobble) occurs at 5’ end of anticodon

83
Q

Wobble base-pairing rules

A 
The genetic code is degenerate because:
1. More than one tRNA is attached to the same AA
2. Wobble base-pairing  - More than one codon is  recognized by the same anticodon
5'       3'
G       U or C
A        U
C        G
U        A or G
I          U,C or A
84
Q

Ribosomal RNA

A

Ribosomes are composed one small and one large subunit
Each subunit is composed of 1-3 rRNA types and up to 50 proteins
23S and 28S rRNA function as ribozymes – formation of peptide bonds
Prokaryote 70s, 50s and 30s
Eukaryote 80s, 60s and 40s

85
Q

Ribosome structure

A

Ribosome is a molecular machine that allows the tRNA anticodon to interact with mRNA codons
Binding site for mRNA within the small subunit
Three binding sites for tRNA that bridge both the small and large subunits A site/Aminoacetyl site: Binds incoming aminoacyl-tRNA (tRNA nwith an attached AA)
P site/Peptidyl site Binds peptidyl-tRNA (tRNA with an attached peptide)
E/Exit site binds the deacylated tRNA prior to release

86
Q

Steps involved in translation initiation in prokaryote

A

Ribosomes are assembled during the initiation of translation
Initiation involves the recruitment of the 30S subunit + initiation factors (IF1 and IF3) to the mRNA
IF3 prevents 30S and 50S subunits from associating
IF1 blocks A site
Initiator codon (AUG) is correctly positioned at the P site to Sets translational reading frame
Shine-Dalgarno sequence pairs with the 3’ end of the 16S rRNA in 30S subunit
IF2 recruits the initiator tRNA (tRNAmeti) to the P site
Methionine attached to initiator tRNA is formylated – N-formylmethionine (fMet)
All E.coli proteins contain fMet at the start (N-terminus) of the protein
IFs released and large 50S subunit associates with the initiation complex
Aminoacyl site is now able to receive charged tRNAs

87
Q

Steps involved in translation elongation in prokaryote

A

Charged tRNA associate with elongation factor Tu (EF-Tu) – called a ternary complex
Only a ternary complex that has an anticodon complementary to the codon will enter the A site
Ribosome changes shape and EF-Tu leaves the tRNA
An amino end (in the A site) and a carboxyl end (in the P site) are now in close proximity in the peptidyltransferase centre
Within the peptidyltransferase centre:
bond between the AA and tRNA in Psite broken
peptide bond between AAs in Pand A is catalysed by the 23S rRNA
Elongation factor G (EF-G) displaces the peptidyl tRNA from the A site
mRNA moves through the ribosome in a 3’ → 5’ direction
Uncharged tRNA enters E site
EF-G leaves ribosome
Uncharged tRNA leaves E site
A site is empty and can now accept the next changed tRNA
Elongation in eukaryotes very similar: EF-Tu = eEF1aand EF-G = eEF2

88
Q

Steps involved in translation termination in prokaryote

A

Elongation adds amino acids at 15-20 aa/sec - until a STOP (nonsense) codon is encountered
Release factors (RF1/2) recognize stop codons Similar in structure to the tRNA ternary complex
No charged tRNA in peptidyltransferase centre
RF1 recognizes UAA or UAG
RF2 recognizes UAA or UGA
Water molecule in the A site – release of polypeptide from P site
RF3 promote removal of RF1/2
Ribosome subunits disassociate
RF1 = eRF1 in eukaryotes, binds to all three stop codons, eRF3 promotes polypeptide release

89
Q

Transcription and translation are coupled in bacteria

A

Lack of a nuclear compartment in bacteria

Bacterial translation begins before transcription has terminated

90
Q

Translation initiation in eukaryotes

A

mRNA bound by eukaryotic Initiation Factor (eIF4) complex
Cap-binding protein eIF4E recruits other eIF4 factors
eIF4F has helicase activity – removes 2ndstructure around the initiator codon
Eukaryotic mRNAs are exported from the nucleus (transcription/translation are sequential)
Subsequently translated as circular molecules in the cytoplasm eIF2 binds directly to initiator tRNAMeti– recruited to the 40S ribosomal subunit
Small ribosomal subunit and eIFs to form a 43S pre-initiation complex along with the initiator tRNAMeti in the cytoplasm
eIF4 complex facilitates binding of the 43S pre-initiation complex to mRNA – 48S pre-initiation complex formed 40S ribosome subunit moves along the mRNA
Unwinds secondary structure and scans for the initiator codon AUG

91
Q

Different in initiation of translation in prokaryote and eukaryote

A

No Shine-Dalgarno sequence
Ribosome recruited to mRNA through protein-protein interactions
Typically uses the first AUG encountered

92
Q

Kozak sequence

A

Sequences around the AUG determines whether it will be selected as an initiator codon
Kozak consensus sequence defines the optimal initiation site for most eukaryotic mRNAs
Purine at upstream
G behind UAG

93
Q

Translation elongation in eukaryotes

A

Alignment of initiator codon in P site of 40S leads to recruitment of eIF5
eIF5 dislodges eIF4 complex, 60S subunit recruited to 40S subunit
80S functional ribosome complex formed
Elongation commences

Foundational genetic (15 decks)

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