(L1,2,3) Gene Expression, Transcription and translation

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Model systems
E.coli, mice, Saccharomyces cerevisiae, Humans, Fruit flies
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Why is gene expression important?
It makes sure that there is the correct amount of protein in the right place at the right time
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What does gene expression require?
Raw materials, energy source, genetic information
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What are differences between organisms due to?
Differences in gene products and patterns of gene expression
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What process involves the copying of DNA to RNA?
Transcription
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What enzyme carries out transcription? (Transcribes DNA)
RNA polymerase
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What RNA molecule is made as a result of transcription?
Messenger RNA
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What does the base sequence of mRNA provide?
Information to make proteins with specific amino acids
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What process is carried out by ribosomes?
Translation
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Deoxynucleotide
Sugar (deoxyribose), Phosphate and a base
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What charge does a phosphate molecule have?
Negative
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What is the difference between ribose and deoxyribose?
Deoxyribose lacks the oxygen molecule on the 2' carbon atom
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What direction does a DNA molecule have?
5' end to 3' end
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How many hydrogen bonds are between the base pairs?
2 between A-T and 3 between G-C
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What are the DNA strands known as?
Antiparallel
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Describe the double helix structure of DNA
2nd strand is reversed, 3' end to 5' end, this is the antisense strand where transcription occurs, it is also know as the template strand
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What sequence does the mRNA molecule have?
The same base sequence as the sense strand (5'-3') but instead of T it has U
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What are the bonds between amino acids?
Peptide bonds
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What determines the sequence of amino acids in a polypeptides sequence in translation?
The sequence of bases in the mRNA
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What is a triplet codon?
3 bases coding for an amino acid
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What is tRNA?
A small RNA molecule that had an amino acid bound to it (aminoacyl-tRNA), it recognises the corresponding bases on the mRNA
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What is a start codon?
Specifies the beginning of translation, AUG for methionine
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What is a stop codon?
Specifies end of translation, UAA, UGA, UAG
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Describe the structure of tRNA
Unique sequence of modified bases, "clover leaf" secondary structure, Amino acids bind covalently
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What in the tRNA binds to the codon in mRNA?
Anticodon (sequence of bases on tRNA on the anticodon loop)
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What causes the imperfect "wobble" base pairing of tRNA?
When tRNA can recognise more than one codon (can occur at the third position)
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Name the keys to specificity
Base pairing, The genetic code (3 bases coding for an amino acid), tRNA specificity, Aminoacyl-tRNA synthase (joins amino acid to corresponding tRNA)
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Describe the chemically modified tRNA's?
Some tRNA molecules can have a chemically modified base called inosine (I) at the corresponding position in the anticodon, can form 2 hydrogen bonds to U, C and A
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Name the binding sites on a ribosome
mRNA binding site, Site-E, Site-P and Site-A
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Describe the initiation process of translation
Methionyl-tRNA (eukaryotes) Formylmethionyl-tRNA (bacteria) binds to P-site in small subunit, mRNA binds to binding site on small subunit, start codon binds to anticodon on Met-tRNA, Large subunit of ribosome binds, ready for elongation
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Describe the elongation process of translation
Aminoacyl-tRNA for next amino acid recognises bases and binds in A-site, peptide bond formed between AA on P-site, polypeptide chain transferred to A-site, large subunit moves, resets ribosome so old tRNA expelled via E-site, new tRNA on P-site
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Describe the termination mechanism of translation
Translation ends at first 'in-frame' stop codon, release factor protein binds to A-site instead of tRNA, peptide-tRNA bond is hydrolysed, releasing new polypeptide, subunits dissociate
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Where does the energy for translation come from?
Hydrolysis of GTP
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Describe the first GTP hydrolysis in translation
EF1 (Eukaryotes) EF-Tu (Bacteria) carries aminoacyl-tRNA and GTP molecule to ribosome, if base paring is correct, EF releases the tRNA and hydrolyses GTP to GDP+P and energy
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Describe thee second GTP hydrolysis in translation
EF2 (Eukaryotes) EF-G (Bacteria) is bound with GTP to the A-site, it hydrolyses GTP and resets the ribosome ready for the next aminoacyl-tRNA
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What is the one gene, one enzyme hypothesis?
Each gene controls the production, function and specificity of a particular enzyme
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What is the genome?
The whole of the DNA of an organism (The set of genes and non-coding sequences)
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How many genes do bacteria typically have?
~4000
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How many genes do animals typically have?
~25,000
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What is a promotor?
Provides a binding site for RNA polymerase in transcription, and specifies a start point and direction of transcription
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What specifies where transcription ends?
The transcription terminator
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How are genes generally transcribed in eukaryotes?
Individually
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How are genes generally transcribed in bacteria?
Many genes are arranged into operons (clusters of genes), all genes in an operon are transcribed from one promoter to give polycistronic mRNA, genes in operons tend to have related functions
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Name some issues with translation
1) Methionine is found in the middle of proteins as well as at the beginning, 2) In an operon, translation must start at more than one place in an mRNA molecule
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What do these problems mean?
The cell must be able to distinguish start codons from other AUG's
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Describe the structure of RNA polymerase core enzyme
5 polypeptides- 2 alpha copies, 1 beta, 1 beta primed, 1 omega
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The core enzyme can synthesise RNA but what can it not do?
Recognise specific promoter sequences
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What does specific binding to promoters require?
A sigma factor (binds specifically to promoters)- Core enzyme + Sigma factor --> holoenzyme
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What is a holoenzyme?
An enzyme which can recognise -35 and 10 sequences
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Describe the most common bacterial transcription termination
mRNA fold into a stem loop structure, followed by a succession of U, The structure destabilises RNA-DNA interaction, RNA polymerase dissociates from DNA and transcription terminates
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It makes sure that there is the correct amount of protein in the right place at the right time

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What does gene expression require?

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Card 4

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What are differences between organisms due to?

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Card 5

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What process involves the copying of DNA to RNA?

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