Genetics Chapter 10

8/13/2019 Genetics Chapter 10

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Gene Expression:

Transcription

Chapter 10

• RNA is an intermediate between DNA and

protein

• types of RNAs

• the process of transcription

• posttranscriptional modifications of RNAs

Central Dogma

red dashes :

post 1958

Types of RNA• Messenger RNA (mRNA)

• Transfer RNA (tRNA)• Ribosomal RNA (rRNA)

• Small nuclear RNA (snRNA)

• microRNA (miRNA)

Evidence :

• need an intermediate between DNA (nucleus) and protein

synthesis (cytoplasm) in eukaryotes

• mRNAs direct protein synthesis

• nucleotide sequences of mRNAs are complementary to DNAs

RNA is intermediate between

DNA and protein :

RNAs that are important for

translation

mRNA tRNA rRNA

How was the role of RNA initially

determined?

Nucleotide Composition

• RNAs produced by organisms have

base ratios similar to organism’s DNA

DNA-RNA Hybridization

• Strands that are complementary can

base pair with each other

DNA-RNA Hybridization

Double-stranded DNA

Denatured DNA

+ complementary

DNA-RNA

hybridization1. cytoplasmic RNA with nuclear

2. virus DNA and infected

bacterial RNA

2 Differences between DNA and RNA

RiboseDNA:

Deoxyribose

Uracil

Thymine

• Does not usually form a double helix

• Usually transcribed from one strand of the DNA

Noncoding DNA strand (template) : complementary to mRNA

Coding DNA strand (nontemplate) : same sequence as mRNA (with U for T)

note : A ≠ U

G ≠ C

RNA Transcription

• Using RNA polymerase, makes RNA using DNA as a template

• ** Transcription is regulated in each cell only certain genes are transcribed in 1 cell at 1 time

allows for regulation of protein synthesis in a cell

• RNA polymerase holoenzyme (E. coli )Five subunits: two subunits

one subunit

one ’ subunit

one subunit ( factor )

- recognizes transcription start sequence

Requirements for RNA polymerase

1. Must identify beginnings and ends of genes

2. Must recognize correct DNA strand

3. Must accurately copy DNA into an RNA copy

Transcriptional Initiation

• Begins at a promoter sequence that RNA polymeraseassociates with before transcription

major site where gene transcription is controlled

• Consensus sequence (in E. coli )

-10 5’ TATAAT 3’ (Pribnow Box, -10 sequence)

-35 5’ TTGTCA 3’ (-35 sequence)

Describes binding site for RNA polymerase

Promoters are ~60 bp long and

composed of consensus sequences

Conserved sequence : identical nucleotide sequences

Consensus sequence : some variation, but high frequency of

certain nucleotides in a group of sequences

(coding strand shown)

DNA Footprinting Identifies

Consensus Sequences

T i ti l i iti ti l ti hi

Transcriptional initiation : relationship

between DNA strands and RNA

-10 +1

consensus

Start of transcript

Promoter of the rrnB gene in E. coli

-35 -10 +1consensus consensus

Start of

transcript

factor binds

- initiates transcriptionfactor binds

-increases transcription

-50consensus

other consensus sequences enhance or inhibit transcription by binding regulatory proteins

and factors can also recognize other regions of different genes (or proteins bound to them)

(not always present)

Transcriptional Initiation in E. coli

1. Sigma ( ) subunit of RNA Polymerase recognizes -10 and-35

2. Holoenzyme (RNA polymerase, ’, subunits)

binds promoter

Closed Promoter Complex

Open Promoter Complex (DNA is unwound)

3. Transcriptional initiation occurs

Closed promoter complex

Open promoter complex

Initiation

Elongation

(5’ 3’ for RNA)

(3’ to 5’ along the DNA)

(sigma factor dissociates)

Transcriptional Termination in E. coli

1. Rho dependent : requires rho ( ) protein + specific

RNA stem-loop structure

2. Rho independent-requires specific RNA stem-loop

structure only

Both have stem loop structure

- causes RNA polymerase to pause

Termination : rho independent

and rho dependent

UUUUU present in rho independent terminator only

Termination : rho independent

and rho dependent

rho independent

rho dependent

because of weak A-U base pairing, RNA

dissociates from DNA

Rho interaction with polymerase causesdissociation of RNA from DNA

Overview of Bacterial Transcription

Initiation:

Elongation:

Termination:

Comparison of prokar otic messenger

Comparison of prokaryotic messenger

RNA (mRNA) and its DNA

• Open Reading Frame (ORF) : translatable segment, gene product

– AUG start codon

– UGA, UAA, UAG stop codons (nonsense codons)

– contains termination sequence but not promoter

• 5’ untranslated region (5’ UTR) : leader sequence that is not translated

• 3’ untranslated region (3’ UTR), sequence after stop codon that is not translated

- 5’ and 3’ UTRs regulate stability of mRNA and have roles in translation

ribosomal RNA

(rRNA)

Ribosomes : sites of protein synthesis

Composed of protein + rRNA :

50S subunit : 34 proteins+ 23S, 5S rRNA

30S subunit : 21 proteins, 16S rRNA

One transcript for all three rRNAs

23S rRNA + 16S rRNA + 5S rRNA

rRNAs : not translated ; no ORF, no start or stop codons, no UTRs

- catalyze peptide bonds during translation

S : Svedberg unit

- rate of sedimentation

in sucrose density gradient

transfer RNA

(tRNA)

Transfer RNA (tRNA)

aminoacyl tRNA synthetase :

attaches specific aa to tRNA

Similarities among tRNAs :

i. all ~80 nt long ; not translated, no ORF, no start or stop codons, no UTRs

ii. all have a cloverleaf shape : acceptor stem, T loop, D loop, anticodon loop

iii. modified bases : help to form loops (do not base pair)

ribothymidine

dihydrouridine

Modified Bases in tRNAs

Processing of tRNA Transcript

Removal of 3’ and 5’ ends

Add 3’ CCA

(eukaryotes),

remove introns

Modify specific

P k ti d E k ti

Prokaryotic and Eukaryotic

Transcription

Similarities :• both use promoters to indicate initiation of transcription

• ribonucleotides (inlcuding uracil) are used to make transcript

• transcription moves 5’ 3’

Differences :

• number of different RNA polymerases

• complexity of promoter sequences

• localization of transcription and translation

• modifications to mRNAs after transcription

Eukaryotic Transcription

RNA polymerases:

RNA polymerase I: transcribes most rRNA genes

28S, 18S, 5.8S rRNAs

RNA polymerase II: transcribes mRNAs, most snRNAs,and miRNAs

RNA polymerase III: transcribes small genes such

as tRNAs, 5S rRNA, U6 snRNA

** all 3 enzymes cannot bind DNA themselves; recruited

prokaryotes :

only 1 RNA polymerase and primase ; both bind DNA directly

1. UBF Binds the upstream promoter element,helps other proteins bind core promoter

Eukaryotic RNA Polymerase I Promoter

2. TATA Binding Protein (TBP) Binds DNA, bends

(**positions RNA polymerase)

(binds A-T region

nonspecifically)

3. RNA Polymerase I Binds to TBP

Transcription preinitiation complex

3 T f E k ti

3 Types of Eukaryotic

RNA Polymerase III Promoters

55-80 bp downstream;

bind TFIIIA and/or TFIIIC

** binding of TFIIIC allows TFIIIB to bind near initiation site

binds TFIIIB

binding of other factors here

enhances TFIIIB binding toTATA

Two Types of Eukaryotic RNA

Polymerase II Promoters

initiator region downstream

promoterelement- these genes are usually transcribed by pol II in all cells throughout development

TATA Bi di P t i b d th DNA

TATA Binding Protein bends the DNA

when it binds

Eukaryotic RNA Polymerase II

• Does not bind to DNA directly (similar to pol Iand III)

• Requires general transcription factors to

associate with the DNA :1. TBP binds to TATA box

2. TBP associated factors (TAFs) bind to

generate preinitiation complex3. RNA polymerase binds to promoter

4. RNA polymerase is phosphorylated

Initiation of

Transcription:

Preinitiation

Complex(subunit of TFIID)

Initiation of Transcription Acti ator

Initiation of Transcription: Activator

Proteins Bind Enhancers

enhancers can be upstream

or downstream of promoter

- activators bind, ↑ transcription

silencers are often upstream

of promoters

- repressors bind, ↓ transcription

**Tissue specific gene expression :

due to binding of transcription factors

to specific promoters

DNA bends

to bind

activator/enhancer

to polymerase

Regulation of Transcriptional

Initiation

Combinatorial Control

• RNA polymerase II complex contains proteinsthat link activators/repressors to core enzyme

• modular assembly of different factors on a

basal transcription machinery

- allows for controlled transcription of specific

genes in certain cells at a particular time

Initiation of Transcription:

1. Preinitiation complex

2. Phosphorylation of RNA polymerase

3. DNA strands are separated4. RNA polymerase begins to transcribe

Initiation of Transcription

(primer : 10-12 bp)

Transcriptional Elongation

• 5’ to 3’• * Disruption of histones (HATs) in chromatin

• Proofreading

rate : ~60 nucleotides/second !

Proofreading by RNA Polymerase II

stimulated by TFIIS

extrusion of short region

of RNA

Eukaryotic DNA Transcription

Transcription

Differences :

• eukaryotes : transcription occurs in nucleus,translation in cytoplasm

- prokaryotes : transcription and translation is“coupled”, both occur in the cytoplasm

• eukaryotes : promoters more complex (transcriptionfactors)

• eukaryotes: posttranscriptional modification of mRNA

• # of different RNA polymerases (eukaryotes: 3,

prokaryotes: 1)

Coupled Transcription and

Translation in Prokaryotes

PostranscriptionalModifications :

1. modify 5’ end

2. modify 3’ end

3. remove intervening sequences

Post Transcriptional Modifications in

Eukaryotic ribosomes : RNA and protein

Post Transcriptional Modifications inEukaryotes – ribosomal RNAs

http://biosiva.50webs.org/translation.htm

1. Processing of ribosomal transcript

2. Base modification by small nucleolar ribonucleoprotein particles

(snoRNPs) : small RNAs and proteins

- uridines pseudouridines

- methylated riboses

Post Transcriptional Modifications inEukaryotes – ribosomal RNAs

rRNA is transcribed by RNA polymerase I in multiple copies

Post Transcriptional Modifications inEukaryotes – RNA polymerase I

Eukaryotes – RNA polymerase II

RNA polymerase II transcripts:

5’ CAP

3’ poly A tail

Intron removal

important for :

1. export from nucleus

2. mRNA stability

3. translation initiation

5’ CAP

functions of cap :

1. protects mRNA from 5’3’ degradation

2. needed for removing 1st intron

3. needed for mRNA export from nucleus

4. ribosome recognizes and binds – needed for translation

3’ polyA Tail

3 polyA Tail

• RNA is cut by endonuclease

downstream from AAUAAA

• polyA tail (15-250 adenosines)

added by polyA polymerase

Importance :

1. required for removal of last intron

2. helps mRNA to be translated

3. improves mRNA stability (protects from nucleases)

Intron

Removal

introns : interveningDNA sequences that

do not code for protein

• transcribed into preRNA,

but are removed before

RNA is translated

• present in mRNA, rRNA,

and tRNA(prokaryotes: tRNA, rRNA only)

exons : sequences

present in mature RNA

E1 I1 E2 I2 E3

E id f P f I t i DNA

Evidence for Presence of Introns in DNA

DNA-mRNA

hybridization

Intron Removal (in vitro): Self splicing

Group I Intron

Intron acts as an enzyme : ribozyme

Group II Intron

Intron acts as an enzyme : ribozyme

Intron Removal: Self splicing

Group II Intron

Lariat structure

in intron

Intron Removal in Eukaryotes (in vivo):

Protein Mediated

Spliceosome : protein-RNA complex required for removal of intron- composed of snRNPs (U1,U2,U4,U5,U6)

(small nuclear ribonucleoproteins)

Consensus sequences required:

• 5’ end of the intron

• 3’ end of the intron

• within intron: lariat branch point

Intron Removal: Sequence of

1. U2AF binds 3’ end and Py-rich tract

2. U1 binds 5’ end ; U2 base pairs with branch point

3. U4, U5, U6 cause release of U1 and bring two exons

together

4. U4 is released, intron lariat generated

5. U5 facilitates release of lariat and ligation of the two

Intron Removal: Sequence of Steps

• over 98% of introns undergo this splicing reaction

Advantages of Introns

Advantages of Introns• Exon shuffling over time to produce novel

gene products : variation• needed for mature mRNA export : reduce

production of nonfunctional protein

• ** alternative splicing: produce more thanone gene product from one gene

rat -tropomyosin

Splicing Repressors

Prevent recognition of 3’ splice junction, forces next 3’

junction to be used

Splicing Enhancers

Promotes recognition and use of poorly recognizedintron-exon junctions

Alternate Splicing for Gene with

Two Promoters

Trans-Splicing

p g Attaches 5’ end of one mRNA to different mRNA

(seen mostly in worms, some human genes)

RNA Editing

• Guide RNA Editing- guide RNA provides complementary

sequence for mRNA RNAs base pair

- RNA is cleaved by endonuclease

- nucleotide is inserted

- RNA ligase closes the break

• Nucleotide Substitution

- deamination of specific bases

(possible if sequence of DNA is slightly different than sequence of encoded protein)

RNA Editing

Guide RNA

editing

RNA Editing through

Deamination of Specific Bases

(changes mRNA codon)- used by enzyme AID

(on DNA) in somatic

hypermutation of Ig gene

- can introduce a STOPcodon (UAA)

- can alter the function of a

glutamate receptor

microRNA

Result : Gene silencing

1. Translation of target mRNA blocked and/orendonuclease degradation of RNA

2. Transcript can be blocked in nucleus byfostering a repressive chromatin structure

microRNA (miRNA)

(RNA Induced Silencing Complex)

delete loop, incorporate 1 strand into RISC

Revised Central Dogma

RNA tumor viruses,HIV

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