PROT SYNTH & REG OF GENE EXPR.ppt

8/14/2019 PROT SYNTH & REG OF GENE EXPR.ppt

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PROTEIN SYNTHESIS &REGULATION OF GENE

EXPRESSIONAbdul Salam M. Sofro

Faculty of Medicine & LPPM YARSIUniversity JakartaRC Biotechnology UGM Yogyakarta


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Teaching aims

By the end of the lecture:

students are expected to understand

the molecular or biochemical processesof DNA replication, transcription andprotein synthesis

Students are expected to understandthe principles of gene expression


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Core topics

Overview

DNA Structure & replication of DNA Transcription of DNA (RNA synthesis)

Protein synthesis (translation)

Regulation of gene expression


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Overview

Protein biosynthesis is also called translation(translation of information from four-letterlanguage & structure of nucleic acid into 20-letter language & structure of protein)

This process requires:

Informational mRNA exported from nucleus

bilingual tRNA that reads the message

Ribosomes that serve as catalytic &organizational centers

A variety of protein factors & energy


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Cont.

Polypeptide/proteins are formed bysequential addition of amino acids in thespecific order determined by info

carried in the nucleotide sequence ofthe mRNA

Proteins are often matured orprocessed by a variety of modifications

Levels of translation is regulated


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Cells vary in their need & ability to

synthesized proteins: Growing cells & dividing cells must

synthesize much larger amounts of

protein Some cells synthesize proteins forexport as well as for their own use

(e.g. liver cells synthesize largenumbers of enzymes needed for theirmetabolic pathways as well as proteinsfor export including serum albumin)


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Terminally differentiated (adult) redblood cells have no nuclei, do notdivide & do not synthesize proteinsdue to the absence of components ofthe biosynthetic apparatus


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Components of the translationalapparatus

mRNA Ribosomes

tRNA


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mRNA

Carrier of information present in DNA In eukaryotes (including human) usually

are synthesized as larger precursor

molecules that are processed prior toexport from the nucleus

It has several identifying

characteristics: almost always monocistronic(encodinga single polypeptide)


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5 end is capped with 7-methylGfollowed by non-translated region whichmay be short or up to a few hundrednucleotides in length separated the capwith translational initiation signal (AUG)

Uninterrupted sequences that specify aunique polypeptide sequence; followedby 3-untranslated sequence, usually

about 100 nucleotides in length, beforeterminated by a 100-200 nucleotide longpoly-A tail


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In prokaryotes:

5 terminus is not capped Mostly polycistronic (encoding several

polypeptides & include more than one

initiation AUG sequence) Ribosome positioning sequence is

located about 10 nucleotides upstream

of a valid AUG initiation signal An untranslated sequence follows the

termination signal, but no poly-A tail


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tRNA

A bilingual translator molecule

All tRNA molecules have several commonstructural characteristics (3-terminal CCA

sequence to bind amino acid, a highlyconserved cloverleaf secondary structure &L-shape three dimensional structure)

Great specificity in interaction with mRNA &the aminoacyl-tRNA synthetase


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Transfer of genetic information

Replication of DNAtransmission of genetic informationfrom parental cell to its daughter cells

Transcription of DNAtransmission of genetic informationfrom DNA to RNA

Translation of RNA(polypeptide/proteinbiosynthesis)transmission of genetic information

from RNA to polypeptide/protein


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DNA structure & Replication of

DNA


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DNA structure & Replication of DNA

DNA is a macromolecule that ultimatelycontrols every aspect of cellularfunctions through protein synthesis

DNA is a transforming agent as well asmaterial responsible for transmittinggenetic information from one generation

to the next

DNA RNA Protein


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Human Genome Size

NUCLEAR GENOME* 23 pairs of chromosomes 2x ( 3 x 109 b.p) 2 meters

DNA / Cell* 2 x ( 3 x 1012cells)meters DNA in human body8,000 x earth to moon

MITOCHONDRIAL GENOME* circular, 16,569 bp


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DNA (deoxyribonucleic acid)

Sugar is deoxyribose

DNA is a polymer ofdeoxyribonucleotides

Bases are adenine (A), guanine (G),cytosine (C) and thymine (T)

Double strands anti parallel


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Needs RNA primer

Produces : Leading strand of new DNA

(complementary to old DNA template

with free 3-OH end) Lagging strand of new DNA with

Okazaki fragments (complementary to

old DNA template with free 5- end


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Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html
http://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.html


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Image Source: http://esg-www.mit.edu:8001/esgbio/dogma/repl.html
http://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.html


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Transcription of DNA

(RNA synthesis)


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RNA (ribonucleic acid) Sugar is ribose RNA is a polymer of ribonucleotides.

Bases are adenine, guanine, cytosine anduracil (instead of thymine)

Single strand

Three types of RNA: mRNA, tRNA & rRNA


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Transcription of DNA (RNA synthesis)

In chromosomes, DNA acts as a templatefor the synthesis of RNA in a processcalled transcription:

Only one strand of DNA act astemplate (35 strand)

Originated from any point of DNA of

the gene (Polypeptide gene, tRNA geneor rRNA gene) at the promotor site

Does not require RNA primer


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Involved:

RNA polymerase NTP (ATP, GTP, CTP, UTP)

Termination signal

In most mammalian cells, only 1% of theDNA sequence is copied into a functionalRNA (mRNA). Only one part of the DNA

is transcribed to produce nuclear RNA,and only a minor portion of the nuclearRNA survives the RNA processing steps.


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Promoter

Bind RNA polymeraseprotect DNAfrom digestion

Two common motifs on 5 : -10 sequence5-TATAAT-3 and -35 sequence (6 bplong) 5-TTGACA-3

At coding strand = sense (+) strand &template strand = antisense (-) strand


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Strong vs. weak promoter (every 2 sec& once in 10 min.)

Specific sequences near it influencedby regulatory proteins & interact withRNA polymerase

Recognized by sigma subunit RNApolymerase (2holoenzyme)


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RNA polymerase

Searches DNA for initiation site There are many more molecules of RNA

polymerase per cell than DNA polymerase.

RNA polymerase proceeds at a rate muchslower than DNA polymerase(approximately 50-100 bases/sec for RNA

versus near 1000 bases/sec for DNA the fidelity of RNA polymerization is much

lower than DNA


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Unwinds a short stretch of double-helical

DNA to produce DNA template Select correct dNTP & catalyses formation of

fosfodiester bond

Interact with activator & repressor proteinthat modulate the rate of transcription

Unwinds nearly two turns of template DNAbefore initiating RNA synthesis

Starts with pppG or pppA

Primers are not needed


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DNA template

Transcription bubble for elongationcontaining RNA pol, DNA, nascent RNA

Form RNA-DNA hybrid helix (about 12bp long/one turn of A-DNA)

Direct RNA synthesis

Transcribed by RNA pol (lack nucleaseactivity) with lower fidelity than that ofreplication (error rate 1 in 104or 105)


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Nascent RNA & processing

Undergo little or no modification for mRNA(maybe translated while being transcribed)

Cleaved & modified for rRNA & tRNA in Ecoli, a primary RNA transcript is excised togenerate three rRNAs (5S, 16S & 23S) & onetRNA by ribonuclease P

May contain arrays of several kinds of tRNAs

or several copies of same tRNA Addition of nucleotides to termini of some

RNA chains (CCA to 3 tRNA)

Modifications of bases & ribose units of rRNAs


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Transcription termination

Formation of fosfodiester bonds ceases RNA-DNA hybrid dissociates Melted DNA region rewinds

RNA pol releases DNA Precisely controlled Stop signals in DNA template regions e.g.

palindromic GC-rich region followed byAT-rich region forms RNA hairpinstructure

Rho protein helps terminate transcription


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One of the most

important stages inRNA processing isRNA splicing. Inmany genes, the

DNA sequencecoding for proteins,or "exons", may beinterrupted bystretches of non-coding DNA, called"introns".
http://en.wikipedia.org/wiki/Image:Proteinsynthesis.png


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In the cell nucleus, the DNA thatincludes all the exons and introns ofthe gene is first transcribed into acomplementary RNA copy called"nuclear RNA," or nRNA.

In a second step, introns are removedfrom nRNA by a process called RNAsplicing. The edited sequence is called"messenger RNA," or mRNA.


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Protein synthesis

(Translation of mRNA)


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The ribosome moves from codon tocodon along the mRNA. Amino acids areadded one by one, translated intopolypeptidic sequences dictated by DNA

and represented by mRNA. At the end, arelease factor binds to the stop codon,terminating translation and releasing the

complete polypeptide from the ribosome.


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Codon

Three-letter code words ( a triplet code) Unambiguous

Non-overlapping

Without punctuation

Universal

Can be found either in DNA (sensestrand) and mRNA

The collection of codons (64) makes up the genetic code


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Threenonsensecodons (UAA,UAG, UGA) donot code forspecific aminoacid and areutilized astermination

signal


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A = adenine G = guanine C = cytosineT = thymine U = uracil

DNA transfers information to mRNA in theform of a code defined by a sequence ofnucleotides bases.During protein synthesis, ribosomes movealong the mRNA molecule and "read" itssequence three nucleotides at a time(codon) from the 5' end to the 3' end.
http://www.accessexcellence.org/RC/VL/GG/protein_synthesis.htmlhttp://www.accessexcellence.org/RC/VL/GG/protein_synthesis.html


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Each amino acid is specified by the

mRNA's codon, and then pairs with asequence of three complementarynucleotides carried by a particular tRNA

(anticodon). Since RNA is constructed from fourtypes of nucleotides, there are 64

possible triplet sequences or codons(4x4x4).


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Three of these possible codons specify

the termination of the polypeptidechain. They are called "stop codons(nonsense codons). That leaves 61

codons to specify only 20 differentamino acids. Therefore, most of theamino acids are represented by morethan one codon. The genetic code is

said to be degenerate.


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Amino acids specified by each codon sequence on mRNA

Ala: Alanine Cys:Cysteine

Asp: Asparticacid

Glu: Glutamicacid

Phe:

Phenylalanine

Gly: Glycine His: HistidineIle:

Isoleucine

Lys: Lysine Leu: LeucineMet:

Methionine

Asn:

Asparagine

Pro: ProlineGln:

GlutamineArg: Arginine Ser: Serine

Thr:

Threonine

Val: ValineTrp:

Tryptophane

Tyr: Tyrosisne

P t i t sl ti t k s l b th


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Protein translation takes place by thefollowing steps

1. Formation of the initiation complex

2. Elongation of the polypeptide chain (onerepetition of the steps a, b and c for everyamino acid incorporated into the protein beingmade):

a. binding of aminoacyl-tRNA

b. peptide bond formationc. translocation

3. Termination


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Remember !

Proteins are polypeptides made up ofindividual amino acidslinked together,

Carbohydrates are polysaccharidesmade up of individual monosaccharideslinked together, and

Nucleic acids arepolynucleotides made

up of individual nucleotides linkedtogether.
http://www.ncc.gmu.edu/dna/amino.htmhttp://www.ncc.gmu.edu/dna/amino.htm


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http://www.med.unibs.it/~marchesi/pps97/course/section4/prtsynth/page5.html


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http://www.med.unibs.it/~marchesi/pps97/course/section4/prtsynth/page6.htmlhttp://www.med.unibs.it/~marchesi/pps97/course/section4/prtsynth/page7.html


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Single base changes in DNA sequence

followed by changes in mRNA molecules mayhave one of several effects whentranslated into protein:

No detectable effectsilent mutation

Missense effectmissense mutation

Appearance of nonsense codon that result

in premature termination of polypeptidechain being synthesizednonsensemutation

S b tit ti f i id i t i


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Substitution of amino acids in proteincauses missense mutations (illustration on

Hemoglobin molecule): Acceptable missense mutations

Hb Hikari: AAA or AAG (lys) to AAU or AAC

(asp) Hb E: GAA or GAG (glu) to AAA or AAG (lys)

Partially acceptable missense mutations

Hb S: GAA or GAG (glu) to GUA or GUG (val) Unacceptable missense mutations

Hb M: Hb (Fe2+) to met Hb (Fe3+)


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Frameshift mutations result fromdeletion or insertion of nucleotides generates altered mRNAs

May be one, two, three or multiplesnucleotides


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Regulation of gene expression


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In bacteria & viruses: Alteration of gene expression isrequired by organism to adapt to

environmental changes

involvesinteraction of specific binding proteinswith various regions of DNA in theimmediate vicinity of the transcription

start site


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In eukaryotes:

in addition to those proteins,alteration of gene expression alsoinvolves tissue specific expression;regulation by hormones, metals &chemicals; gene amplification; generearrangement; posttranscriptional

modification


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Type of responses to a regulatory signal

Type A: increased rate of geneexpression that is dependent upon thecontinued presence of inducing signal

Type B: increased rate of geneexpression that transient even in thecontinued present of regulatory signal

Type C: increased rate of geneexpression that persist indefinitely evenafter the termination of the signal

Type of gene regulation


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Type of gene regulation

Positive regulation: The expression of genetic info isquantitatively increasedby the presence

of a specific regulatory element (themolecule is positive regulator)

Negative regulation:

the expression of genetic info isdiminishedby the presence of a specificregulatory element (the molecule isnegative regulator)

Model of gene expression in prokaryotes


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Model of gene expression in prokaryotes

One cistron, one subunit concept insteadof one gene one enzyme concept (cistronis the smallest unit of gene expression,coding for the structure of the subunitof a protein molecule)

Inducible genes: their expressionincreases in response to an inducer

Constitutive genes: their expression isreasonably constant (not known to besubject to regulation)


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The earliest level of regulation is at DNA level during transcription


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Legend:


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Process of creating a hybrid strand of DNA/RNA

The two strands of a DNA molecule are denaturedby heating to about

100C = 212F (a to b). At this temperature, the complementary base pairs

that hold the double helix strands together are disrupted and the helix

rapidly dissociates into two single strands.

The DNA denaturation is reversible by keeping the two single stands

of DNA for a prolonged period at 65C = 149F (b to a). This process is

called DNA renaturationor hybridization.

Similar hybridization reactions can occur between any single strandednucleic acid chain: DNA/DNA, RNA/RNA, DNA/RNA. If an RNA transcript

is introduced during the renaturation process, the RNA competes with the

coding DNA strand and forms double-stranded DNA/RNA hybrid molecule

(c to d).

These hybridization reactions can be used to detect and characterizenucleotide sequences using a particular nucleotide sequence as a probe.
http://www.accessexcellence.org/RC/VL/GG/dna_molecule.htmlhttp://www.accessexcellence.org/RC/VL/GG/dna_molecule.html

PROT SYNTH & REG OF GENE EXPR.ppt

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