prok & euk guided summary 2015

7/23/2019 Prok & Euk Guided Summary 2015

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Guided Summary Y6 2015

Last updated by Ms. Emeline Choo, Mr. WY Ngan, Mr Ariff Chan 1

A. CORE SYLLABUS

(4) Organisation of Prokaryotic and Eukaryotic Genome (I)

Learning outcome 4 (a)Compare the structure and organization of prokaryotic and eukaryotic chromosomes.

Key words:Double-stranded DNA, encodes gene products, Genes, Promoter, association with histone, level ofcondensation, nucleoid, Nucleus, Origin of replication, Introns, Exons, Enhancers, SilencersCentromere, Telomere, genes organized into operons

Similar i t ies:

1. Prokaryotic and eukaryotic chromosomes are both made of dou ble stranded DNA* andencodes gene products essential for the function of the organism;

2. They genetic information they carry are organized into genes* where each gene is controlledby a promoter* upstream;

Differences:

Pt of comparison Prokaryote Eukaryote

1. Size andappearance ofchromosome

Prokaryotes have a single,small chromosome

Eukaryotes have multiple, largechromosomes;

2. Association withhistones

Prokaryotic chromosomes arenot associated with proteins

Eukaryotic chromosomes areassociated with histone* proteins;

3. Level ofcondensation

Prokaryotic chromosomes havea low level of condensationforming supercoiled loops

Eukaryotic chromosomes have avery high level of condensationforming multiple levels of coiling;

4. Location Prokaryotic chromosomes arefound in the nucleoid region Eukaryotic chromosomes arefound in the nuc leus* ;

5. Origin of replication Prokaryotic chromosomes havea single or igin o f repl icat ion*

Eukaryotic chromosomes havemultiple or igin of repl icat ions* ;

6. Presence of non-coding sequences

Non-coding sequences are notcommon in prokaryotechromosome

Non-coding sequences are verycommon in eukaryotechromosomes making up to 98%of the DNA;

7. Presence of introns Prokaryote chromosomes donot have in t rons*

Eukaryote chromosomes havein t rons * interspersed betweencoding exons *;

8. Gene regulatoryelements Promoters* regulate theexpression of genes inprokaryotes

Besides prom oters, enhancers* as well as si lencers* areinvolved in gene-regulation ineukaryotes;

9. Repeatedsequences

Repeated sequences are rarein prokaryotes

There is one centromere* perchromosome while the ends ofthe chromosome has telomeres* ;

10. Operons Related genes are oftenorganized into operons* ;

Operons* are rare in eukaryoticchromosomes;





Comments:

Please make sure that the sentences in each cell under prokaryotes and eukaryotes columns are complete sentences.You can also write in continuous prose instead of a table if you are fluent enough and have a clear idea about what your point of comparison is. Prokaryotes is ……. while eukaryotes is………e.g. Prokaryotes have a single, small chromosome while eukaryotes have multiple, large chromosomes;

Learning outcome 4 (b)Describe the structure and function of introns, promoters, enhancers and silencers.

Key words:Non-coding sequences, exons, coding sequences, splicing, introns excised, alternative splicingcombinations of exons, pre-mRNA, mature mRNA, upstream of transcriptions start site, RNApolymerase, critical elements, consensus sequence, frequency of initiation of transcription, pribnowbox, TATA box, enhancer regulatory element, specific transcription factors, activators, transcriptioninitiation complex, looping of DNA, silencer, repressor

Introns:

Structure1. Introns are non-coding sequences found within a gene;2. They are interspersed between exons* which are coding sequences;3. During spl ic ing* , introns are excised out while exons are joined together;

Function

4. Having introns allow al ternat ive sp l ic ing* to take place where different segments of DNA arespliced out while combining different combinations of exons;

5. Resulting in a single pre-mRNA* yielding multiple mature mRNAs or one gene yieldingmultiple proteins;

Promoters:

Structure1. Promoters are non-coding sequences found just upstream of the transcription start site of agene;

Function2. They serve as recognition sites for the binding of RNA polym erase* ;3. The closer the sequences of the cr i t ical elements* resemble the consensus sequence, the

stronger the promoter and the higher the frequency of initiating transcription;4. E.g. Pr ibnow box * of prokaryotes and TATA box * of eukaryotes;

Enhancers:

Structure

1. Enhancers are non-coding regulatory elements that has an influence on the expression of agene far away from them;Function

2. They bind to specific transcription factors called act ivators* which increases the frequency oftranscription;

3. by promoting the assembly of the t ranscr ipt ion ini t iat ion com plex* as they cause the loopingof DNA that brings the activators, RNA polymerase and general transcription factors togetherat the promoter;





Silencers:

Structure

1. Silencers are non-coding regulatory elements that has an influence on the expression of agene far away from them;

Function

2. They bind to specific transcription factors called repressors* which decreases the frequencyof transcription;3. by preventing the assembly of the t ranscr ipt ion ini t iation com plex *;

Comments:

For each item, please address both structure and function.

Learning outcome 4 (c)Describe the role of telomeres and centromeres.

Key words:

Non-coding DNA, without loss of genetic information, protect and stabilize terminal ends ofchromosomes, chromosome breaks, apoptosis, fusion of chromosome ends, base pair with thetemplate on telomerase, proper alignment of telomerase, allows for its own extension, constrictedregion along the chromosome, sister chromatids to adhere, allow attachment of kinetochore proteins,spindle fibers, alignment of chromosomes, separation of chromosomes.

Telomeres:

1. Telomeres are non-coding tandemly repeating DNA sequences at the terminals of linearchromosomes;

2. Allow successive rounds of DNA replication and consequent shortening of daughter

chromosome molecules without (loss of genetic information/erosion of genes);3. By forming a loop with the 3’ overhang, they protect and stabilize terminal ends of

chromosome;4. which prevents the ends of chromosomes from being recognized as chromosome breaks

which can lead to (cell death/apoptosis);or prevent fusion of ends of different chromosomes;

5. Possess an overhang which base pairs with the template on telomerase so as to ensureproper alignment of telomerase;

6. hence allows for its own extension in a repeated manner;

Centromere:

7. Centromeres are non-coding, tandemly repeating DNA sequences found on a constrictedregion along the chromosome;

8. They allow sister chromatids to adhere to each other and also allow attachment ofkinetocho re proteins* which in turn attach to spindle fibers;

9. This is important in enabling alignment of chromosomes along the metaphase plate and alsoseparation of chromosomes during anaphase;





Learning outcome 4 (d)Describe the process and significance of gene amplification in xenopus oocyte.

Key words:Upregulation of gene expression, increases gene copy number, increases templates for transcriptionrapid demand, cannot be met by transcription and translation of a single gene, rRNA gene cluster,

ribosomes, protein synthesis, rapid growth of oocyte, extrachromosomal circular DNA, rolling circlemechanism, nick, free 3’ end for continuous strand synthesis, recircularises, template used to formcomplementary strand.

Signi f icance:

1. Gene amplification allows the upregulation of gene expression by increasing the gene copynumber of a specific gene(s) and hence the templates used in transcription;

2. This is to meet the rapid demand for ribosomes that cannot be met by transcription andtranslation of a single gene;

3. The gene amplified in Xenopus oocyte is the rRNA gene cluster* as these genes code for animportant component of ribosomes;

4. that are important in protein synthesis during the rapid growth of the oocyte;

Process:

5. The genomic chromosome give rise to extrachrom osom al circular DNA* carrying the rRNAgene cluster;

6. From this first ring many more copies of circular DNA is synthesized through the rol l ing c irc le

mechanism* ;7. A nick occurs in one strand of the circular DNA, and using the free 3’ end, continuous DNA

synthesis occurs while the 5’ end is displaced; 8. Another nick is made to release the displaced strand that recircularises and act as a template

to form the complementary strand;





(4) Organisation of Prokaryotic and Eukaryotic Genome (II)

Learning outcome 4 (e)Describe the eukaryotic processing of pre-mRNA in terms of intron splicing, polyadenylation and 5capping.

Key words:introns excised, exons joined together, spliceosomes, adenosine monophosphates, 3’ end, poly-Apolymerase, 3’ poly-A tail, 7 methyl-guanosine, 5’ end

Intron sp l ic ing:

1. Splicing is a process whereby in t rons * are excised and exons * are joined together and iscarried out by spl iceosomes *.

2. Spliceosomes recognize the points of excision at the intron-exon boundaries.

Polyadenylat ion:

3. Polyadenylation is a process whereby multiple adenosine monophosphates are added to the 3end* the pre-mRNA by poly-A polymerase *, forming a 3’ poly -A tail *;

5 ’ capping: 4. 7 methyl-guanosine is added to the 5’ end * of the pre-mRNA.

Learning outcome 4 (f)Define control elements and explain how control elements (e.g. promoter, silencer and enhancers)and other factors (e.g. transcription factors, repressors, histone modification and DNA methylation)influence transcription.

Key words:

non-coding DNA sequences, transcription factors, promoter, silencer, enhancer, upstream ofthe transcription start site, general transcription factors, usually located far away from a gene,specific transcription factors, activators, repressors

Promoter, binding of RNA polymerase, critical elements, consensus sequence, stronger thepromoter, higher the frequency of initiating transcription, pribnow box, TATA box

silencers, specific transcription factors, repressors, decreases frequency of transcription,looping of DNA, prevent assembly, general transcription factors, RNA polymerase, promoter,transcription initiation complex is not formed

enhancer, specific transcription factors, activators, increases frequency of transcription,

looping of DNA, promote assembly, general transcription factors, RNA polymerase,promoter, transcription initiation complex is formed

Control elements:

(what are control elements?)

6. Control elements are non-coding DNA sequences that t ranscr ipt ion factors * bind toregulate transcription;

7. Control elements include promoter *, si lencer * and enhancer *.





8. A promoter usually lies upstream of the t ranscr ipt ion star t si te * and binds to general

t ranscr ipt ion factors *;9. Enhancer and silencer are usually located far away from a gene and bind to speci f ic

transcr ipt ion factors * called activators * and repressors * respectively;

(how do control elements influence transcription?)

Promoter:10.Promoter * serve as the recognition site for the binding of RNA polym erase* ;11. The closer the resemblance between the sequences of the cri t ical elements* in the

promoter and the consensus sequence, the stronger the promoter and the higher thefrequency of initiating transcription;

12. e.g. Pr ibnow b ox* of prokaryotes and TATA b ox* of eukaryotes;

Silencer (only eukaryotes):

13.Silencers * bind to specific transcription factors called repressors* which decreases thefrequency of transcription;

14. Looping of spacer DNA * allows repressors bound at silencers to

15. Prevent assembly of general transcr ipt ion factors * and RNA polymerase * at thepromoter * and the t ranscr ipt ion ini t iat ion com plex * is not formed.

Enhancer (only eukaryotes):

16.Enhancers* bind to specific transcription factors called act ivators* which increases thefrequency of transcription;

17. Looping of spacer DNA * allows activators bound at the enhancers to18. promote assembly of general transcr ipt ion factors * and RNA polymerase * at the

promoter * and the t ranscr ipt ion ini t iat ion comp lex* is formed.

Key words: Acetylation, deacetylation, histones, decondensation, condensation, chromatin, histone

acetylases, add acetyl groups, removes positive charges on histones, removes positivecharges on histones, decreases the electrostatic interactions, negatively charged DNA,adding acetyl groups, general transcription factors, RNA polymerase, bind the promoter, formthe transcription initiation complex, remove acetyl groups, restores positive charges onhistones, increasing the electrostatic interactions, negatively charged DNA, positivelycharged histones, prevents binding, general transcription factors, RNA polymerase,promoter, transcription initiation complex is not formed.

DNA methylation, addition of methyl group, cytosine, condensation of chromatin, preventtranscription, blocking binding, general transcription factors, RNA polymerase, promoter,

preventing the formation, transcription initiation complex, recruiting DNA-binding proteins,condense chromatin

Other factors:

(how do other factors influence transcription?) Transcription factors:

19.(refer to above section “how do control elements influence transcription?”)

Histone modification:





20.Acetylat ion * and deacetylation * of histones * can result in decondensation andcondensation of chromatin * respectively;

21.Histone acetylases * add acetyl groups to lysine residues on histones and removespositive charges on histones *;

22. This thereby decreases the electrostatic interactions between negatively charged DNA *and the histones;

23. This allows general transcr ipt ion factors * and RNA polymerase * to bind the promoter *to form the t ranscr ipt ion ini t iat ion comp lex *, to allow transcription to occur;24.Histon e deacetylases * remove acetyl groups from histones and restores positive charges

on histones *;25. This increases the electrostatic interactions between negatively charged DNA * and the

positively charged histones *;26. This prevents binding of general transcr ipt ion factors * and RNA polymerase * to the

promoter * and the t ranscr ipt ion ini t iation com plex * is not formed, and transcription isprevented.

DNA methylation:

27.DNA methylases * adds methyl group to selected cytos ine * nucleotides in e.g. a CGsequence;28. This blocks binding of general transcr ipt ion factors * and RNA polymerase * to the

promoter *, preventing the formation of the t ranscr ipt ion ini t iat ion complex * andtranscription does not occur;or

29. This recruits DNA-binding proteins (e.g. repressors, histone deacetylases, repressivechromatin remodeling complexes) to condense chromatin;

30. such that general transcr ipt ion factors * and RNA polym erase * to cannot bind thepromoter * to form the t ranscr ipt ion ini t iation com plex *, transcription does not occur;





Learning outcome 4 (g)State the various ways in which gene expression may be controlled at translational (e.g. half life ofRN A, 5’ capping, initiation of translation) and post-translational level (e.g. biochemical modificationand protein degradation).

Learning outcome 4 (h)Outline the differences between prokaryotic control of gene expression with the eukaryotic model.

Genomic level Prokaryotic

None – prokaryotes (in general) don’t rely on modifying chromosomal structures to control geneexpression.

Eukaryotic

(1) Organisation of chromosome

What happens to expression of gene X if it is found in a region of chromosome calledheterochromatin or euchromatin?

Heterochromat in* = DNA winds more tightly around histonesprevents access of general transc ription factors * and RNA polym erase* to promote

of gene, t ranscr ip t ion in i t ia t ion comp lex * not formed at promoter* inactive gene expression / no transcription

Euchromat in * = DNA winds less tightly around histones promotes access of general transc ription factors * and RNA polym erase * to promote

of gene, t ranscr ip t ion in i t ia t ion comp lex * formed at promoter* active gene expression / transcription

What is X chromosome inactivation?

1 of 2 X chromosomes in mammals like female humans is compacted to become Barbody.

Describe what happens to expression of gene X if it is found on a chromosome thaundergoes X chromosome inactivation.

Compact structure of Barr body prevents access of RNA polymerase, and generatranscription factors to promoter of gene inactive gene expression / no transcription

(2) DNA methylation

What is DNA methylation?

Addition of a methyl group to selected cytos ine * (C) nucleotides

Describe what happens to expression of gene X when it is methylated.

General transcription factors * and RNA polymerase * cannot bind to promotert ranscr ip t ion in i t ia tion c omp lex * is not formed at promoter* no gene expression

(3) Chromatin remodelling complex

What is chromatin remodelling complex?

A complex of proteins capable of altering structure of nucleosomes

Describe what happens to expression of a gene when chromatin remodelling complexcauses DNA to bind more tightly/loosely to histones.

When DNA is more tightly coiled around histones, general transcr ip t ion factors * andRNA polym erase * cannot bind promoter * and t ranscr ip t ion in i t ia t ion c omp lex * is noformed. no transcription

When DNA is less tightly coiled around histones, general transc ription factors * and RNA

polymerase * can bind promoter * and t ranscr ip t ion in i t ia tion complex * is formed. transcription can occur





(4) Histone deacetylation / acetylation

What is histone deacetylation?- Removal of acetyl groups from lysine residues of histones by histo ne deacetylases*

Describe what happens to expression of gene X when histones are deacetylated.- Removal of acetyl groups restores positive charge of histones *, increasing the

electrostatic interactions between negatively charged DNA * and histones- Prevent binding of general transcr ip t ion factors * and RNA polymerase *, t ranscr ip t ion

in i t iat ion comp lex * is not formed at promoter * of gene.- Transcription prevented.

What is histone acetylation?- Addition of acetyl groups to lysine residues of histones by histo ne acetylases*

Describe what happens to expression of gene X when histones are acetylated.- Addition of acetyl groups removes positive charge of histones *, decreasing the

electrostatic interactions between negatively charged DNA * and histones- Allow binding of general transcription factors * and RNA polym erase *, t ranscr ip t ion

in i t iat ion comp lex * is formed at promoter * of gene.- Gene is expressed / transncribed

(5) Gene amplification

Define gene amplification.

Describe what happens to expression of gene X when the region it is contained inundergoes gene amplification.

Replication of a specific gene to create more copies of that gene.

Gene of interest exists in high copy number, so increased copies of its mRNA and proteinformed.

Prokaryotic Eukaryotic

Transcriptionallevel

What is transcription?(Must include terms: DNA, mRNA, tRNA, rRNA, transcribed, RNA polymerase)

Transcription is a process in which base sequence of a gene (DNA) is used as a template todirect synthesis of RNA (mRNA, tRNA, rRNA). Need RNA polymerase (enzyme) andtranscription factors (proteins).

Where does transcription take place?Prokaryotes: Nucleoid Eukaryotes: Nucleus

What does transcriptional control mean?Controlling when and how often a gene is transcribed to form mRNA.





Transcriptional control is the most important partof control of gene expression for prokaryotes.

(1) Promoter- What is a promoter?

Promoter = DNA sequence where RNA

polymerase * bind to start/initiatetranscription.

- What are the critical elements?Critical elements = short DNA sequenceslocated within the promoter & consists of: -10 sequence (aka Pribnow box) -35 sequence

- What is the role of the -10 and -35sequences in regulating frequency ofgene expression?The more similar the -10 and -35sequences are to the consensus

sequences *, the stronger the promoter *,the higher frequency of transcription /gene expression.

(2) Sigma factor- What are sigma factors?

Sigma factor = a subunit of RNApolymerase that recognises the -10 & -35sequences in prokaryotic promoters.Sigma factor + core RNA polymerase =RNA polymerase holoenzymeSigma factor binds to core RNApolymerase to form the RNA polymeraseholoenzyme which scans along the DNAfor the -10 and -35 sequences.

- How do sigma factors regulate frequency

of transcription? Different sigma factors recognisedifferent promoters. Availability of sigma factors will determinewhich genes are transcribed and thefrequency of transcription (e.g. highlevels of sigma factor that recognisespromoter of gene X high transcriptionfrequency of gene X).

(3) Operon- What is an operon?

Genes with related functions aregrouped together. Expression of these

genes are controlled by one commonpromoter * and transcribed into asingle, polyc is tron ic mRNA *.

- What is the advantage of groupinggenes that code for gene products withrelated functions together?Since expression of the genes in theoperon are controlled by one promoter,all the genes in the operon are turnedon and off together, hence moreefficient.

(A) Promoter- What is a promoter?

Promoter = DNA sequence where RNA

polymerase * and general transcr ip t ion

factors * bind to form the t ranscr ip t ion

in i t ia t ion complex * to start/initiatetranscription.

- What are the critical elements?Critical elements = short DNA sequenceslocated within the promoter & consists of: TATA box at -25 site CAAT and GC boxes

- What is the role of the TATA box, CAAT& GC boxes in regulating frequency ofgene expression?- TATA box determines the preciselocation of the transcription start site

- CAAT & GC boxes help to recruitgeneral transcription factors * andRNA polymerase * to promoter forassembly of the t ranscr ip t ion

in i t iat ion c omplex *. The greater the similarity between

the critical elements and theconsensus sequences *, thestronger the promoter *, the higherfrequency of transcription / geneexpression.

(B) Enhancers / Silencers

What are enhancers & silencers?Enhancer = DNA sequence that bind tospeci f ic transcr ip t ion factors * calledactivators * to increase transcription

frequency.Silencer = DNA sequence that bind tospeci f ic transcr ip t ion factors * calledrepressors * to prevent transcription.

Where are enhancers & silencerslocated?Usually far from a gene - thousands ofnucleotides upstream / downstream of thegene, but can also be found within intron.

Describe how do enhancers influence theexpression of a gene.- Act ivators * bind to enhancer* - Spacer DNA bends and allows

activators bound to enhancer- to promote binding of genera

transcr ip t ion factors * and RNA

polymerase * to promoter * to form thet ranscr ip t ion in i t iat ion complex *.

- Bound activator may recruit histone

acetylases * or chrom at in remod el ling

complexes * to increase accessibility topromoter.





(4) Operator & Repressor- How do repressors influence frequency

of transcription of genes?Repressor * (protein) bind to operator *(in lac and trp operon) preventbinding of RNA polymerase * topromoter * no transcription / basallevel of transcription.

(5) CAP binding site & CAP- How does Catabolite Activator Protein

(CAP) influence frequency oftranscription of genes?CA P * (protein) bind to CAP bind ing

si te * (in promoter of lac operon) increases affinity of RNA polym erase *for promoter * higher frequency oftranscription.

Describe how do silencers influence theexpression of a gene.- Repressors * bind to si lencer* - Spacer DNA bends and allows

repressors bound at silencer region - to prevent binding of genera

transcr ip t ion factors * and RNA

polymerase * and t ranscr ip t ion

in i t iat ion c omplex * is not formed atpromoter *.

- Bound repressor may recruit histone

deacetylases * (HDACs) or repressive

chrom at in remodel l ing com plexes * todecrease accessibility to promoter, orinterfere with action of activators boundto enhancer.

Post-

transcriptionallevel

None – prokaryotes (in general) don’t modify

mRNA after transcription. mRNA is used directlyfor translation.Often, both transcription and translation happenat the same time. As the mRNA is beingsynthesised, ribosomes begin binding to mRNAto synthesise polypeptides.

As opposed to prokaryotes, transcription and

translation do not occur concurrently. Why? Presence of nuclear envelope * in

eukaryotes prevents transcription andtranslation from taking place concurrently

In eukaryotes, pre-mRNA * is formed fromtranscription and needs to undergo pos t -

t ranscr ip t iona l modi f icat ion * to formmature mRNA * before translation can takeplace.

Post-transcriptional modification of mRNA

What is the order of the 3 modifications?(a) capping at 5’ end

(b) splicing of pre-mRNA(c) adding a poly- A tail to the 3’ end

(polyadenylation)

Where do these modifications take place inthe cell?Nucleus

For each of the 3 modifications:

Describe each modification and itssignificance to gene regulation(a) Capping at 5’ end of pre-mRNA

Add a 7-methyl guanosine (modified

guanosine) to 5’ end of pre-mRNA.Significance?

─ helps cell to recognise mRNA from otherRNAs for splicing & polyadenylation

─ helps mRNA to exit from nucleus tocytoplasm for translation

─ protects pre-mRNA from rapiddegradation by cellular ribonucleasesIncreases half life of mRNA.

─ recognised by translation ini t iat ion

factors * which then help smal





ribosomal subunit bind to mRNA so thattranslation can occur

(b) Splicing of pre-mRNAProcess where in t rons * are excisedexons * are joined together byspl iceosome *.Significance?- cut out non-coding introns or else a

protein will not be properly made.- Alternative spl icing *; all in t rons *

excised, different combination of exons *are joined together to give differentmature mRNA* allows 1 gene to code for differentpolypeptides.

(c) Polyadenylation at 3’ end of pre-mRNAProcess of adding adenosinemonophosphates to 3’ end * of pre-mRNAby poly-A polym erase *.

Significance?- Slow down degradation of mRNA by

ribonucleases. Enhances half-life /stability of mRNA.

- a signal to direct export of maturemRNA from nucleus to cytoplasm.

- works with 5’ cap* to regulate mRNAtranslational efficiency.

Translational level What is translation?Process by which mRNA is used by ribosomes as a template to synthesise polypeptides.

Where does translation occur?Free ribosomes in cytoplasm or ribosomes on rough endoplasmic reticulum.

What is a polycistronic mRNA? A single mRNA codes for several differentpolypeptides. Contains multiple start and stopcodons in a single mRNA. Only prokaryotesproduce polycistronic mRNA.

(1) Half life of mRNA

What does an mRNA with a short half lifemean?Short half life = mRNA is rapidly degradedby ribonucleases, can’t get that manypolypeptides translated from it.

Describe the 2 ways to control mRNA halflife.Prokaryotic mRNAs inherently have arelatively short half-life as compared toeukaryotes’ due to lack of 5’ cap* & 3’poly-A tai l *. Anti-sense RNA * binds to the mRNA *,block translation / target the mRNA fordegradation shorten half life of mRNA.

Note: eukaryotic mRNAs are usuallymonocistronic and not polycistronic.

(1) Half life of mRNA

What does an mRNA with a short half lifemean?Short half life = mRNA is rapidly degraded byribonucleases, can’t get that manypolypeptides translated from it.

Describe the ways to control mRNA half life.Half-life/ stability is influenced by factors suchas length of 3’ poly-A tai l * and presence of 5cap *.





(2) Start of translation

Describe the 2 ways to control translationinitiation on an mRNA.- A repressor * protein binds at/near toShine-Dalgarno sequence *, preventsbinding of small ribosomal subunit,ribosomes cannot assemble properly

translation fails.- Ant i-sense RNA * binds to the mRNA *,

prevents binding of small ribosomalsubunit, ribosomes cannot assembleproperly

translation fails.

(2) Start of translation

Describe the 2 ways to control translationinitiation on an mRNA.Translation can be blocked by a repressor *protein that binds to these parts on an mRNA:5’ cap* and/or its vicinity i.e. 5’ untranslatedregion * (5’ UTR), and 3’ untranslatedregion * (3’ UTR).

Post-translationallevel

What does post-translational control mean? Has the polypeptide been made?Controlling proteins that are already present in the cell by activating or inhibiting their functions, orregulating their levels

None – prokaryotes (in general) don’t dependon modifying proteins to control their activities.

Briefly describe what these post-translationamodifications are, and how they control thegene expression levels:

(1) Covalent modifications

Newly synthesised protein go throughchanges (e.g. glycosyla t ion *, disu l f ide

bond * formation, cleavage *) to form thefunctional protein.

(2) Phosphorylation / Dephosphorylation Newly synthesised protein go through

phosphory la t ion * (add phosphategroup) or dephosphory la t ion * (removephosphate group) so that they canbecome active/inactive respectively.

(3) Protein degradation Proteins no longer needed will be

degraded by proteasomes *. Ubiqui t in *will be added to such proteins, andproteasomes will recognise and degradesuch proteins.





Learning Outcomes4 (i) Describe the functions of common proto-oncogenes and tumour suppressor genes (limited to ras and p53) and

explain how loss of function mutation and gain of function mutation can contribute to cancer.

proto-oncogenes tumour suppressor genes

Define Normal cellular genes that code for productsthat stimulate normal cell growth and

proliferation.

Proteins coded by proto-oncogenes areinvolved in stimulating normal cell division,and signaling pathways.e.g. growth factors, growth factor receptors,growth signal transduction factors,transcription factors

Normal cellular genes which code forproducts which inhibit cell division and help

prevent uncontrolled cell division.

Products of tumour suppressor genesactivate cell cycle arrest, DNA repair and/orapoptosis (programmed cell death).

Explain consequences ofmutation

When proto-oncogenes mutate, they areknown as oncogenes *.

Mutation results in an increase in activity orincreased amount of a proto-oncogene’sprotein product.

Since proto-oncogenes regulate cell division,any extra gene products coded by proto-oncogenes will cause uncontrolled/excessivecell division.

Mutation results in a non-functional tumoursuppressor gene’s protein product.

Since tumour suppressor genes help inhibitcell division (while repairing a mutation), anymissing gene products from them will causecell division to continue without repairingDNA/without going into apoptosis.

Explain how mutationcan be brought about

(a) Substitution mutation such that the proto-oncogene protein coded for ishyperactive or more resistant todegradatione.g.: a regulatory protein (such as growthfactor encoded by proto-oncogene)

(b) Gene amplification unexpectedly makingmany copies of a chromosomal regioncontaining that proto-oncogene. leads to excessive production of proto-oncogene protein

(c) Chromosomal movement such thatunusual exchange of chromosomes causesa proto-oncogene to be placed under thecontrol of an enhancer leads to increased transcription andmore gene products from proto-oncogene

Virus genome that integrate into humangenome may carry its own enhancer leads to increased transcription andmore gene products from proto-oncogene

Any mutations that cause gene productcoded by tumour suppressor gene to be non-functional.e.g.: point mutations (i.e. substitution /addition / deletion mutation)





Ras gene p53 gene

State if gene is a proto-oncogene or tumoursuppressor gene

Proto-oncogene Tumour suppressor gene

Describe role of thesegene products

Ras gene codes for ras proteins which aresignal transducers.

Activated ras proteins transduce signalswhen growth factor binds to receptor todownstream signaling processes. Thisactivates cell division.

p53 gene codes for a specific transcriptionfactor (p53) that binds to DNA to promotesynthesis of cell cycle-inhibiting proteins

p53 protein can activate genes that are involvedin stopping cell division, repairing DNA andstarting apoptosis.

Link consequences oftheir mutation to cancer

Mutation results in a ras protein that isactive all the time.This leads to increased cell division evenwhen growth factor doesn’t bind to thereceptor. Uncontrolled cell division leads tocancer.

Mutation results in a p53 protein that is notactive or whose amount cannot be increased.This leads to continued cell division even whenDNA is not repaired, accumulating mutations(characteristic of cancer).

Gain-in-function mutation Loss-in-function mutation

Description A mutation that causes a gene to beexpressed in a place/ at a time when it isnot normally expressed, or the geneproduct is hyperactive.

A mutation that causes a gene product to non-functional

Type of genes themutation affects

Proto-oncogene Tumour suppressor gene

Number of alleles thathas to be mutated inorder to be cancerous

One allele need to be mutated- mutation in just one copy is enough togive extra gene products / hyperactivegene products that cause the cell cycle toescape normal control.

Both alleles need to be mutated- if only one allele is mutated, there is stilanother allele that still codes for functionaltumour suppressor gene products to inhibit celdivision.

Characteristics of geneproducts from mutation

Gene products of mutated proto-oncogenes become hyperactive / resistantto degradation, or are produced inexcessive amounts

Gene products of mutated tumour-suppressorgenes are defective / non-functional.

Effect on cell cycle Overstimulate cell cycle Can’t stop cell cycle to repair damages.Cells with accumulated mutations keep dividing.





Learning Outcomes

4 (j) Describe the development of cancer as a multi-step process.

Definition:Cancer is a group of diseases characterised by uncontrolled cell division and spread of abnormal cells.

Multi-step development of cancer The development of cancer requires the accumulation of mutat ions * in the genes which control regulatorycheckpoin ts * of the cell cycle in a single cell

This will disrupt the normal cel l cycle *, thus causing the cell to undergo excessive cell growth andproliferation

A gain-in-function * mutation is a dominant mutation where mutation in just one copy/allele of a proto

oncogene * will result in its overexpression which will result in the production of excessive amounts of growthfactors, or production of hyperactive/degradation-resistant growth factors, leading to cell proliferation

A loss-of- function * mutation is a recessive mutation where mutations in both copies/alleles of a tumour

suppressor gene * will disrupt their ability to inhibit cell cycle, enable DNA repair and promote apoptosis

Upregulation/activation of the genes coding for te lomerase * result in telomeres being lengthened and thecell can thus dividing indefinitely as the chromosomes are prevented from shortening with each DNAreplication cycle.

Loss of contact inh ib i t ion * will enable the cells to grow into a tumour/mass of cells.

Angiogenesis * must occur within the tumour so that the blood vessels formed can transport oxygen andnutrients for its growth.

Finally the cells must have the ability to metastasise *. i.e leave the primary site and spread to other tissues

in different parts of the body via the blood stream and form tumours there.

The above mutations should occur for cancer to develop.

As it takes years to accumulate these mutations, developing cancer increases with age.





Characteristics Normal cells Cancerous cells

Cell division Controlled cell proliferation Excessive cell proliferation

Contact inhibition Contact inhibition monolayer of cells

No contact inhibition multiple layer of cells

Ability to differentiate Can differentiate Undifferentiated

Susceptibility to

apoptosis

Undergoes apoptosis Not susceptible to apoptosis

Ability to adhere to othercells

Cell adhesion formation of tissues and organs

Can detach from surrounding cells

Ability to stimulategrowth of blood vessels

No new blood vessels Stimulates growth of new blood vessels withintumours

Pre-requisitebefore canceroccurs

Description

All changes mustoccur in a single cell

Cancer begins with a single cell which undergoes uncontrolled cell division All changes such as getting genetic mutations, not repairing those mutations must all

happen to that same, single cell first.Mutations are not berepaired

Cells naturally contain proof-reading mechanisms to check, and to correct geneticmutations.

Cells with mutationsare not be killed

Cells with genetic damages that can’t be repaired are induced to die (so that damagingmutations are eliminated)

Regulatorycheckpoints must bedisrupted

Regulatory checkpoints in cell cycle prevent a cell from deteriorating into a cancerousone.For a cell to become cancerous, the genes at each checkpoint have to be mutated suchthat they are unable to carry out their normal functions.

prok & euk guided summary 2015

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