biochem lecture handout

8/3/2019 Biochem Lecture Handout

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Other Bases

• Less common basescan occur

• Principally but notexclusively, in transferRNAs

Nucleosides

• Nucleoside: sugar + base

• D-ribose or 2-deoxy-D-ribose covalently bonded to anucleobase by a -N-glycosidic bond

• Lacks phosphate group

Nucleotides

• Nucleotide: PO4 + sugar +base

• a nucleoside + phosphoric acid

• phosphoester bond with an -OH of the sugar, most commonlyeither the 3’-OH or the 5’-OH

• Name based on parentnucleoside with a suffix“monophosphate”

Nucleic Acids

• Polymerization ofnucleotides leads tonucleic acids.

• Linkage is repeated

• (3’,5’-phosphodiesterbond)



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Nucleic Acids

• Levels of structure• 1°structure: the order of bases on the

polynucleotide sequence; the order of bases

specifies the genetic code• 2°structure: the three-dimensional conformation

of the polynucleotide backbone• 3°structure: supercoiling• 4°structure: interaction between DNA and

proteins

DNA - 1° Structure

Deoxyribonucleic acids, DNA:

• Primary Structure: the sequence of bases along the sugar-phosphodiester backbone of a DNA molecule

• base sequence is read from the 5’ end to the 3’ end • System of notation single letter (A, G, C and T)

P P P P3’ O

H

P5’

3’

5’

A C G T G5’

A C G T G3’ or P O

H

DNA - 2° Structure

• Secondary structure: theordered arrangement ofnucleic acid strands• the double helix model

of DNA 2°structure wasproposed by James

Watson and FrancisCrick in 1953

• Double helix: • two antiparallel

polynucleotide strands arecoiled in a right-handedmanner about the sameaxis

• structure based on X-Raycrystallography

T-A Base Pairing

• Base pairing is complementary

• A major factor stabilizing the double helix is base pairing byhydrogen bonding between T-A and between C-G

• T-A base pair comprised of 2 hydrogen bonds



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G-C Base Pair

• G-C base pair comprised of 3 hydrogen bonds

Forms of DNA 2o Structure

• B-DNA

•considered the physiological form•a right-handed helix, diameter11Å

•10 base pairs per turn (34Å) ofthe helix

•occurs in nature• A-DNA

•a right-handed helix, but thicker than B-DNA

•11 base pairs per turn of the helix•has not been found in vivo

• Z-DNA

• a left-handed double helix• may play a role in gene

Other Features of DNA

• Base stacking

• bases are hydrophobic andinteract by hydrophobicinteractions

• in standard B-DNA, eachbase rotated by 32° compared to the next

DNA – 3o Structure in Prokaryotes

Tertiary structure: the 3-D arrangementof all atoms of a nucleic acid;• commonly referred to as supercoiling

• Circular DNA: a type of double-stranded DNA in which the 5’ and 3’

ends of each strand are joined byphosphodiester bonds

• Supercoiling- Further coiling andtwisting of DNA helix.



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DNA – 3o Structure in Prokaryotes

• Supercoiling- Further coiling and twisting ofDNA helix.

• Topoisomerases- an enzyme that relaxessupercoiling in closed circular DNA• Class I: cut the phosphodiester backbone

of one strand, pass the end through, andreseal

• Class II: cut both strands, pass some ofthe remaining DNA helix between the cutstrands, and reseal

• DNA gyrase: a bacterial topoisomerase II

Super DNA Coiled Topology

• Double helix can be considered to be a 2-stranded,right handed coiled rope

• Can undergo positive/negative supercoiling

Supercoiling (3o Structure) in Eukaryotic DNA

• Histone: a protein, particularly rich inthe basic amino acids Lys and Arg;found associated with eukaryotic DNA• five main types: H1, H2A, H2B, H3,

H4

• Chromatin: DNA molecules woundaround particles of histones in abeadlike structure

• Each “Bead” is a nucleosome• Nucleosome consists of: DNA

wrapped around histone core

Denaturation of DNA

• Denaturation: disruption of 2o structure• most commonly by heat denaturation (melting)• double helix unwinds when DNA is denatured

• Renaturation

• double helix can be re-formed with slow cooling and annealing



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DNA REPLICATION

Bidirectional Replication

Prokaryotic DNA Replication

• Replication involves• separation of the two original

strands• synthesis of two new

daughter strands using theoriginal strands as templates

• Semiconservativereplication: each daughterstrand contains one templatestrand and one newlysynthesized strand

Which Direction does Replication go?

• DNA double helix unwinds at a specific point calledan origin of replication

• DNA replication is bidirectional in most organisms;Polynucleotide chains are synthesized in bothdirections from the origin of replication

• At each origin of replication, there are tworeplication forks, points at which new polynucleotidechains are formed

• There is one origin of replication and two replicationforks in the circular DNA of prokaryotes

• In replication of a eukaryotic chromosome, there areseveral origins of replication and two replication forksat each origin



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Replication Fork General FeaturesSummary of DNA Replication inProkaryotes

• Unwinding• DNA gyrase introduces a swivel point

in advance of the replication fork

• a helicase binds at the replicationfork and promotes unwinding

• single-stranded binding (SSB) protein protects exposed regions ofsingle-stranded DNA

• Primase catalyzes the synthesis of RNAprimer

• Synthesis• catalyzed by Pol III

• primer removed by Pol I

• DNA ligase seals remaining nicks

Replication Fork General Features

R N A



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RNA

• RNA

• unbranched chains of nucleotides

• the sugar unit is -D-ribose• Uracil instead of thymine• in general, RNA is single stranded

P P P P3’ O

H

P5’

3’

5’

A C G U G5’

A C G U G3’ or P O

H

Flow of Genetic Information in the Cell

• Mechanisms by which information is transferred inthe cell is based on “Central Dogma”

Information Transfer in Cells

• Information encodedin the nucleotidesequence of DNA istranscribed throughRNA synthesis

• RNA and amino acidsequence then isdictated by DNAsequence

• Central dogma ofbiology



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Transcription

• Overview of Transcription

• RNA is synthesized on a DNA template, catalyzed byDNA-dependent RNA polymerase

• ATP, GTP, CTP, and UTP are required, as is Mg2+

• no RNA primer is required • the RNA chain is synthesized in the 5’ -> 3’ direction;

the nucleotide at the 5’ end of the chain retains itstriphosphate (ppp) group

• RNA polymerase unwinds the helix • the DNA base sequence contains signals for initiation

and termination of RNA synthesis; the enzyme bindsto and moves along the DNA template in the 3’ -> 5’direction

• the DNA template is unchanged

Transcription in Prokaryotes

• E. coli RNA Polymerase:• four different types of subunits: , , ’, and s • the core enzyme is 2’

• the holoenzyme is 2’s • the role of the s subunit is recognition of the

promoter locus; the s subunit is released aftertranscription begins

• of the two DNA strands, the one that serves as thetemplate for RNA synthesis is called the template strand or antisense strand; the other is called thecoding (or nontemplate) strand or sense strand

• the holoenzyme binds to and transcribes only thetemplate strand

Parts of a gene

Promoterregion

Coding region

Promoter region

Termination region

PROKARYOTIC

EUKARYOTIC

The Basics of Transcription

(Sense strand)

(Antisense strand)



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Promoter Sequence

• Simplest of organisms contain a lot of DNA that isnot transcribed

• RNA polymerase needs to know which strand istemplate strand, which part to transcribe, and wherefirst nucleotide of gene to be transcribed is

• Promoter – a DNA sequence that provides directionfor RNA polymerase

Promoter Sequence

σ-subunit initiates strand separation (melting) of the DNA at about -10from the start site.

T G C T A G T C C T G C T A G C C G A T A T A A T G A C A A G A C G T C G A C T T A C A G C G-- - -

A C G A T C A G G AC G A T C G G C T A T A T T A C T G T T C T G C A G C T G A A T G T C G C-- - -

T G C T A G T C C T G C T A G C C G A T A T A AT

A C G A T C A G G AC G A T C G G C T A T A T TA

+1-10

Transcription Start Site

Promoter region



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Chain Elongation (Cont’d) Chain Termination

Two types of termination mechanisms:(1) intrinsic termination- controlled by specific sequences,

termination sites Termination sites characterized by two inverted repeats

Chain Termination (Cont’d)

(2) Termination by rho () protein

• Rho-dependent termination sequences cause hairpinloop to form

Eukaryotic mRNA Splicing



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Characteristic structure of eukaryotic mRNA RNA

• RNA molecules are classified according to theirstructure and function

tRNA

Transfer RNA, tRNA:

• the smallest kind of thethree RNAs

• a single-strandedpolynucleotide chainbetween 73-94nucleotide residues

• carries an amino acid atits 3’ end

• intramolecular hydrogenbonding occurs in tRNA

amino acid

anticodon

rRNA

Ribosomal RNA, rRNA: • found in ribosomes, the site of protein synthesis

• only a few types of rRNA exist in cells• ribosomes consist of 60 to 65% rRNA and 35 to 40%

protein• in both prokaryotes and eukaryotes, ribosomes

consist of two subunits: big and small subunits• particles characterized by sedimentation coefficients,

expressed in Svedberg units (S)• 40S and 60S subunits in eukaryotes• 30S and 50S subunits in prokaryotes

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mRNA

Messenger RNA, mRNA: • carries coded genetic information from DNA to

ribosomes for the synthesis of proteins

• present in cells in relatively small amounts and veryshort-lived

• single stranded

• biosynthesis is directed by information encoded onDNA

snRNA

• Small nuclear RNA (snRNA) is a recentlydiscovered RNA

• Found in nucleus of eukaryotes

• Small (100-200 nucleotides long)

• Forms complexes with protein and form smallnuclear ribonucleoprotein particles (snRNPs)

• snRNPs help with processing of initial mRNAtranscribed from DNA

TRANSLATION:

PROTEIN SYNTHESIS

TRANSLATION

• The sequence of the bases on the mRNAspecifies the sequence of the amino acids in theprotein.

• Transcription takes place in the nucleus, thenthe mRNA is transported to the cytosol

• Translation always takes place in the cytosol,where the mRNA is read and translated at theribosome.

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The Genetic code

• The sequence of aminoacids, derived from thesequence of DNA bases,is specified by the genetic

code, using 4 RNA basesA, U, G, and C taken threeat a time (triplet code).

• There 64 possible “code

words”, called codons

• 1 start codon - AUG• 3 are stop signals:

UAG, UGA, UAA

• 61 specify the 20amino acids withconsiderableredundancy.

The Genetic Code

• Salient features of the geneticcode• triplet: a sequence of three

bases (a codon) is needed tospecify one amino acid

• nonoverlapping: no basesare shared betweenconsecutive codons

• commaless: no interveningbases between codons

• degenerate: more than onetriplet can code for the sameamino acid; Leu, Ser, and Arg,for example, are each codedfor by six triplets

• universal: the same inviruses, prokaryotes, andeukaryotes; the onlyexceptions are some codons inmitochondria

The Genetic Code (Cont’d)

• All 64 codons haveassigned meanings•only Trp and Met have onecodon each

•the third base is irrelevantfor Leu, Val, Ser, Pro, Thr,Ala, Gly, and Arg

•the second base isimportant for the type ofamino acid; for example, ifthe second base is U, theamino acids coded for arehydrophobic

•for the 15 amino acidscoded for by 2, 3, or 4triplets, it is only the thirdletter of the codon thatvaries. Gly, for example, iscoded for by GGA, GGG,GGC, and GGU

Translating the Genetic Message

• Protein biosynthesis isa complex processrequiring ribosomes,mRNA, tRNA, andprotein factors

• Several steps areinvolved

• Before beingincorporated intogrowing protein chain,a.a. must be activatedby tRNA andaminoacyl-tRNAsynthetases

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Shine-Dalgarno Sequence Recognized byE. Coli Ribosomes

T G C T A G T C C T G C T A G C C G A T A T A AT

A C G A T C A G G AC G A T C G G C T A T A T TA

+1

-10

Translation Start Site

A G C U G A A U G U C G C G C C U U U A CA G C …G A UG C C G U C G G U A G C U A AT U A……

met - ser - arg - leu ……………………………………………… - ser

The InitiationComplex

• In all organisms,synthesis of polypeptidechain starts at the N-terminal end, and growsfrom N-terminus to C-terminus

• Initiation requires:• tRNA-fmet – binds to P

site• initiation codon (AUG) of

mRNA• 30S ribosomal subunit• 50S ribosomal subunit• initiation factors IF-1, IF-

2, and IF-3• GTP, Mg2+

• Forms the initiationcomplex

• Step 1• an aminoacyl-tRNAis

bound to the A site• the P site is already

occupied• 2nd amino acid bound to

70S initiation complex;defined by the mRNA

• Step 2• EF-Tu is released in a

reaction requiring EF-Ts

• Step 3• the peptide bond is

formed, the P site isuncharged

• Step 4• the uncharged tRNA is

released• the peptidyl-tRNAis

translocated to the P site• EF-G and GTP are

required• the next aminoacyl-tRNA

occupies the empty A site

Elongation Steps

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Chain Termination

• Chain termination requires• stop codons (UAA, UAG,

or UGA) of mRNA• RF-1 (Release factor-1)

which binds to UAA andUAG or RF-2 (Releasefactor-2) which binds toUAA and UGA

• RF-3 which does notbind to any terminationcodon, but facilitates thebinding of RF-1 and RF-2

• GTP which is bound toRF-3

• The entire complexdissociates setting free thecompleted polypeptide, therelease factors, tRNA,mRNA, and the 30S and50S ribosomal subunits

UAG Simultaneous Protein Synthesis onPolysomes

Post-translational Modification

• Newly synthesized polypeptides are frequently modifiedbefore they reach their final form where they exhibitbiological activity• N-formylmethionine in prokaryotes is cleaved

• specific bonds in precursors are cleaved , as for example,

preproinsulin to proinsulin to insulin• leader sequences are removed by specific proteases ofthe endoplasmic reticulum; the Golgi apparatus then directsthe finished protein to its final destination

• factors such as heme groups may be attached

• disulfide bonds may be formed• amino acids may be modified, as for example, conversion

of proline to hydroxyproline• other covalent modifications; e.g., addition of

carbohydrates

Examples of Posttranslational Modification

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Levels of Protein Structure

1° structure: the sequence of amino acids in apolypeptide chain, read from the N-terminal end tothe C-terminal end

• 2°

structure: the ordered 3-dimensionalarrangements (conformations) in localized regions ofa polypeptide chain; refers only to interactions of thepeptide backbone• e. g., -helix and -pleated sheet

• 3˚ structure: 3-D arrangement of all atoms

• 4˚ structure: arrangement of monomer subunitswith respect to each other

2o Structure: the ordered 3-dimensionalarrangements (conformations) in localized regions of apolypeptide chain; refers only to interactions of thepeptide backbone

α-Helix β-pleated sheet

3o Structure: 3-D arrangement of all atoms

Forces That Stabilize Protein Structure

4o Structure: arrangement of monomer subunits withrespect to each other

Structure of Hemoglobin

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MUTATION

MUTATION - a change in the base sequence of DNA

Origins:

Spontaneous mutation – occurs during normalgenetic and metabolic functions in the cell:

- Replication errors - genetic mispairing

- Base modifications caused by spontaneoushydrolytic reactions

- low frequency: 10-7 to 10-12/generation

Induced mutation – mutagens (increase frequency)

MUTAGENS

• Physical Agents

• UVL – pyrimidine dimers

• Ionizing radiation – x-rays, gamma rays

• Chemical Agents• Nitrous acid

• Intercalating agents

• 5-bromouracil

UVL Ionizing Radiation

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Chemical agentsMUTATION

• Addition• Deletion• Substitution

• Transition• transversion

MUTATION

• Missense mutation• Nonsense mutation• Silent mutation• Frameshift mutation

Mutants

AGU-CGU-GGA-AAU-UGU-CCU-CGA-

ser - arg - gly - asn - cys - pro - arg

AGU-CGU-GCA-AAU-UGU-CCU-CGA-

ser - arg - ala - asn - cys - pro - arg

• Missense mutant

• Base substitution in DNA causes replacementof 1 amino acid residue by another in a protein

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Mutants

• Nonsense mutant

• If base substitution creates a stop codon,thereby terminates protein synthesis

prematurely

AGU-CGU-GGA-AAU-UGU-CCU-CGA- ser - arg - gly - asn - cys - pro - arg AGU-CGU-UGA-AAU-UGU-CCU-CGA-

ser - arg -stop

Mutants

· Silent mutant – - substitution results in triplet coding for the sameamino acid as the original triplet (redundancy ofthe genetic code)

- change usually occurs at the 3 rd base

AGU-CGU-GGA-AAU-UGU-CCU-CGA-

ser - arg - gly - asn - cys - pro - arg AGU-CGU-GGU-AAU-UGU-CCU-CGA- ser - arg - gly - asn - cys - pro - arg

Mutants

• Frameshift mutants

• Due to insertion mutation or deletion mutation

AGU-CGU-GGA-AAU-UGU-CCU-CGA- ser - arg - gly - asn - cys - pro - arg - AGU-CGU-GAG-AAA-UUG-UCC-UCG-A- ser - arg - glu - lys - leu - ser - ser

A

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REPAIR MECHANISMS

• Photoreactivation• Excision repair• Mismatch Repair

LIGHT REPAIR

EXCISION REPAIR OR DARK REPAIR Mismatch Repair in Prokaryotes

• Mechanisms of mismatch repair encompass:

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