Download MBBS Biochemistry PPT 26 Transcription Lecture Notes

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Difference between DNA and RNA synthesis

Chromatin structure and gene expression

Direction of transcription

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Promoter

Transcription unit

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RNA polymerase

Steps of RNA synthesis

Processing of RNA (mRNA, tRNA, rRNA)

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Splicing and clinical implication

Inhibitors

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Case

? A 24 year old man who is being evaluated as a fol ow up to to a preplacement

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medical evaluatio prior to starting his new job.

? He has no significant medical issues. His family history is unremarkable, but he

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knows little of the health status of those family members.

? The physical examination was normal. Routine analysis of his blood included the

following results

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? RBC 4.8x1066/mm3 (4.3-5.9)

? Hb: 9.6g/dl

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? MCV 70microm3 (80-100)

? Serum iron 150 microgram /dl (50-170)

? Based on the data, Hb electrophoresis was performed. The results were as follows

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? HbA 90% (96-98)

? HbA2 6% <3

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? HbF 4% <2

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? What is the possible diagnosis?

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? What is the pathophysiology?

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Classes of Eukaryotic RNA

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Comparison in RNA and DNA synthesis

DNA synthesis

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RNA synthesis

Nucleotide

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dNTP

NTP

Primer

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Yes

No

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Length of the

Entire genome

Portion of the

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genome to be

genome

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copied
Proof reading

Highly effective

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Not highly effective

function
Polarity

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Yes 5' to 3'

Yes 5'to 3'

Base pairing rule

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Adherence

Adherence

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Transcription Unit

? A transcription unit is defined as that region

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of DNA that includes the signals

for transcription initiation, elongation, and

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termination.

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Prokaryotic promoters share two regions of highly

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conserved nucleotide sequence

Promoter: A regulatory region o DNA that serves to bind RNA polymerase

I that in turn binds other substances that wil lead to initiation of transcription

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Adapted from Harper's Biochemistry

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RNA polymerase catalyzes the polymerization

of ribonucleotides

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Adapted from Harper's Biochemistry
Mammalian Nuclear DNA-Dependent

RNA Polymerases

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The transcription cycle

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Adapted from Harper's Biochemistry
bacterial transcription termination signal

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Adapted from Harper's Biochemistry

Transcription control regions in an mRNA-producing eukaryotic gene

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Adapted from Harper's Biochemistry
Transcription elements and binding factors

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Adapted from Harper's Biochemistry

The eukaryotic basal transcription complex

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Adapted from Harper's Biochemistry
Video on transcription

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Some of the Mammalian RNA Polymerase I

Transcription Control Elements, Their Consensus

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Sequences, and the Factors That Bind to Them

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Three Classes of Transcription Factors

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Involved in mRNA Gene Transcription

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RNA polymerase I -mediated mRNA gene transcription is cotranscriptional y

coupled to RNA processing and transport.

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Adapted from Harper's Biochemistry
Modification/processing of mRNA

? Addition of 5' cap

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? In nucleus

? Efficient translation initiation

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? Protection

? Addition of polyA tail (200)

? Cleaved about 20 nt downstream from AAUAA

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recognition sequence

? Poly A polymerase

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? Facilitates translation

? Protection

? Splicing

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SPLICING

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? Spliceosome:

? Involved in converting the primary transcript into

mRNA

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? Consists of

? primary transcript
? five snRNA

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? >60 proteins containing conserved RRM (RNA

recognition ) and SR ( serine arginine) protein motif

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Consensus sequences at splice junctions

PyNPyPyPuAPy

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Adapted from Harper's Biochemistry

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Adapted from Harper's Biochemistry
? Video on splicing

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Mechanisms of alternative processing of mRNA precursors

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Alternative promoter use in the liver

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and pancreatic a-cell glucokinase (GK) genes

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Adapted from Harper's Biochemistry

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Clinical implications of splicing

? Mutation at splice site lead to improper splicing

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? At least 20% of al genetic diseases result of

mutation affecting splicing

? Incorrect splicing of -globin mRNA responsible

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for thalassemia

? Splice site mutation: exons removed and introns

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retained

? Activate cryptic splice site

? In SLE: Antibodies against nuclear protein such as

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Sn RNP

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mRNA Editing

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? Change of Coding information at the mRNA level

? the coding sequence of the mRNA differs from

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that in the cognate DNA

? Example: apolipoprotein B (apoB) gene and mRNA

? In liver, synthesis of a 100-kDa apoB100.

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? In the intestine synthesis of a apoB48

? Cytidine deaminase converts a CAA codon (Glutamine)in the

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mRNA to UAA

? Example: glutamine to arginine change in the

glutamate receptor in trypanosome mitochondrial

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mRNAs

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Processing of rRNA

? generated from 45S long precursor molecules called pre-

rRNAs.

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? 23S, 16S, and 5S rRNA of prokaryotes are produced from a

single pre-rRNA molecule,

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? Eukaryotic 5S rRNA is synthesized by RNA polymerase I I

and modified separately

? pre-rRNAs are cleaved by ribonucleases to yield

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intermediate-sized pieces of rRNA

? In eukaryotes, rRNA genes are found in long, tandem arrays

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? rRNA synthesis and processing occur in the nucleolus, with

base and sugar modifications facilitated by smal nucleolar

RNAs (snoRNA)

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Posttranscriptional processing of

eukaryotic ribosomal RNA by

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ribonucleases (RNases).

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Adapted from Lippincotts Biochemistry

Processing of tRNA

? made from longer precursor molecules

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? Sequences at both ends of the molecule are removed

and intron is removed from the anticodon loop by

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nucleases.

? attachment of CpCpA terminal at the 3 end of the

molecule by the enzyme nucleotidyl transferase.

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? modification of bases at specific positions to produce

the "unusual bases" characteristic of tRNA;

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methylation, reduction, deamination, and rearranged

glycosidic bonds, nucleotide alkylations

? Methylation in the nucleus, whereas the attachment of

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CpCpAOH are cytoplasmic functions

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Biogenesis of micro (mi) and silencing (si)RNAs

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Adapted from Harper's Biochemistry

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Inhibitors of RNA synthesis

Inhibitor

Source

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Mode of action

Rifampicin

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Synthetic

Binds to beta subunit of

derivative of

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RNA polymerase which is

Rifamycin

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inactivated

Alpha amanitin Toxin from

Prevents translocation of

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mushroom

RNA pol II

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During phospho diester

bond formation

3' ?deoxy

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Synthetic

Incorrect entry into chain

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adenosine

analog

causing chain termination

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Summary

? RNA is synthesized from a DNA template by the enzyme DNA

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dependent RNA polymerase

? While bacteria contain a single RNA polymerase (2)

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there are three distinct nuclear DNA-dependent RNA

polymerases in mammals

? RNA polymerases interact with unique cis-active regions of

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genes, termed promoters, in order to form preinitiation

complexes (PICs) capable of initiation. In eukaryotes, the

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process of pol II PIC formation requires, in addition to

polymerase, multiple general transcription factors (GTFs),

TFIIA, B, D, E, F, and H.

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? Transcription exhibits three phases: initiation, elongation, and

termination

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Summary

? The presence of nucleosomes can occlude the binding of both

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transfactors and the transcription machinery to their cognate

DNA cis-elements, thereby inhibiting transcription

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? Most eukaryotic RNAs are synthesized as precursors that

contain excess sequences which are removed-additional

potential steps for regulation of gene expression.

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? All steps--from changes in DNA template, sequence, and

accessibility in chromatin to RNA stability and translatability--

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are subject to modulation and hence are potential control

sites for eukaryotic gene regulation.

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MCQ1

? A 1-year-old male with chronic anemia is found to have

-thalassemia. Genetic analysis shows that one of his

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-globin genes has a mutation that creates a new splice

acceptor site 19 nucleotides upstream of the normal

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splice acceptor site of the first intron. Which of the

following best describes the new mRNA molecule that can

be produced from this mutant gene?

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? A. Exon 1 will be too short.

B. Exon 1 will be too long.

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C. Exon 2 will be too short.

D. Exon 2 will be too long.

E. Exon 2 will be missing

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MCQ2

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? The base sequence of the strand of DNA used as the

template for transcription is GATCTAC. What is the

base sequence of the RNA product? (Al sequences are

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written 5' 3' according to standard convention.)

? A. CTAGATG.

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B. GTAGATC.

C. GAUCUAC.

D. CUAGAUG.

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E. GUAGAUC

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MCQ3

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? A 4-year-old child who becomes easily tired and has

trouble walking is diagnosed with Duchenne muscular

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dystrophy, an X-linked recessive disorder. Genetic analysis

shows that the patient 's gene for the muscle protein

dystrophin contains a mutation in its promoter region. Of

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the choices listed, which would be the most likely effect

of this mutation?

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? A. Initiation of dystrophin transcription will be defective.

B. Termination of dystrophin transcription will be defective.

C. Capping of dystrophin mRNA will be defective.

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D. Splicing of dystrophin mRNA will be defective.

E. Tailing of dystrophin mRNA will be defective.

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MCQ4

? A mutation to this sequence in eukaryotic mRNA

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wil affect the process by which the 3'-end poly-A

tail is added to the mRNA.

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A. CAAT

B. CCA

C. GGGGCG

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D. AAUAAA

E. TATA A A

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