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Foundational genetic > Genes, Genomes and Gene Expression > Flashcards

Genes, Genomes and Gene Expression Flashcards

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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
Key steps in DNA replication – Strand separation
- 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
26
Priming for DNA synthesis
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’
27
DNA synthesis with DNA pol
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
28
Replication in the lagging strand (fill up the gap)
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
29
The replisome
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
30
Initiation of DNA replication in bacteria
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
31
Replication bubbles
Two replisomes are recruited to oriC | DNA replication proceeds in two directions
32
Accessory protein – DNA gyrase
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
33
Segregation of the bacterial chromosome during cell division
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
34
DNA replication in eukaryotes
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
35
The eukaryotic cell cycle
Mitosis -> G1 (Cell growth and preparation for cell cycle entry) -> S ( DNA REPLICATION)-> G2(Cell prepares for the M phase) ->M
36
Initiation of DNA replication in eukaryote
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
37
Linking DNA replication with cell cycle control
DNA synthesis is regulated by the availability of Cdc6 and Cdt1 Cdc6 phosphorylated by Cyclin E-Cdk2 (only found in G1) Cdc6/Cdt1 destroyed
38
Solving the end replication problem - telomerase
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
DNA synthesis at the telomere
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
Telomere cap
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
Chromosomal segregation during cell division (centromer action)
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
Stages of mitosis
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
Classes of RNA
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
Organization of a bacterial protein-coding gene
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
RNA polymerase – how it works in prokaryote strand usage and direction
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
Anatomy of the promoter region in prokaryote
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
Promoter elements – defined by consensus
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
Promoter elements – defined by mutation
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
Bacterial RNA pol structure and role of S factor
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
Steps involved in transcription – initiation in prokaryote
sigma factor positions the RNA polymerase for transcription initiation Holoenzyme recognizes the promoter sequence – closed complex formed
51
Elongation in prokaryote transcription
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
Mechanisms of transcription termination in prokaryote
TWO major mechanisms: Factor-independent termination (Intrinsic/Rho-independent) Rho-dependent termination
53
Factor-independent termination mechanism
•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
Rho-dependent termination mechanism
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
Mechanisms to ensure high levels of transcription in prokaryote
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
Differences between bacteria and eukaryotes transcription (structure and location and processing)
* 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
Eukaryotic RNA pols are different to the bacterial RNA pol
* 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
Roles of different rna pol
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
Organization of a eukaryotic RNA pol II promoter
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
Organization of a eukaryotic RNA pol II promoter: Regulatory elements
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
Requirement for Transcription initiation in eukaryote
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
Steps in transcriptional initiation in eukaryotes
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
Transcription elongation regulated by cis-elements in eukaryote
Mediator enables TFs bound to cis-elements to influence RNA pol II activity
64
Eukaryotic protein-coding genes have introns
- 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
Processing of the pre-mRNA
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
Attachment of the 5’ cap
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
Termination and polyadenylation
§ 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
RNA splicing
Removal of introns from the pre-mRNA and exons joined together – RNA splicing Resulting transcript has a continuous open reading frame
69
Exon-intron boundary
``` 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
Small nuclear RNAs and spliceosome
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
Spliceosome assembly
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
splicing steps
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
Types of alternative splicing
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
Alternative splicing can produce distinct protein isoforms
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
2 process of translation
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
the genetic code of amino acid
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
Codon bias
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
Crick’s adaptor hypothesis:
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
Transfer RNAs
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
Aminoacyl-tRNA synthetase
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
How many aminoacyl-tRNA | synthetases are there in a cell?
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
Some tRNAs recognize more than one codon
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
Wobble base-pairing rules
``` 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
Ribosomal RNA
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
Ribosome structure
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
Steps involved in translation initiation in prokaryote
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
Steps involved in translation elongation in prokaryote
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
Steps involved in translation termination in prokaryote
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
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Transcription and translation are coupled in bacteria
Lack of a nuclear compartment in bacteria | Bacterial translation begins before transcription has terminated
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Translation initiation in eukaryotes
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
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Different in initiation of translation in prokaryote and eukaryote
No Shine-Dalgarno sequence Ribosome recruited to mRNA through protein-protein interactions Typically uses the first AUG encountered
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Kozak sequence
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
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Translation elongation in eukaryotes
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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