Molecular biochemistry

STEP TO PG-MD/MS - DR.AKIF A.B

MOLECULAR BIOCHEMISTRY- I & II

- DR.AKIF A.B

NUCLEIC ACID-Made up of monomer units called Nucleotides.

-Nucleotides are linked to each other by 3’-5’ Phosphodiester bond

-Backbone of nucleic acid = Sugar + Phosphate

NUCLEOTIDES

1) Nitrogen Bases : Purines and pyramidines

2) Pentose sugar

3) Phosphate group

Nucleoside

Beta-N-Glycosidic bond-C1 of Pentose Sugar is linked to N9 of Purines or N1 of Pyramidines

-Most nucleotides are 5’ Nucleotides

-Base sequence og Nucleic Acid is written in 5’-3’ direction

1) At the physiological PH, the DNA molecules are:

A Positively charged.

B Negatively charged

C Neutral.

D Amphipathic

B) Negatively charged

DNA is negatively charged because of phosphate group

2) Nucleoside is made up of (PGI)

A. Pyramidine

B. Histone

C. Purine

D. Sugar

E. Phosphate

C. Purine

D. Sugar

A. Pyramidine

NUCLEOTIDES

1) Nitrogen Bases : Purines and pyramidines

2) Pentose sugar

3) Phosphate group

Nucleoside

3. Apart from occuring in Nucleic acids, Pyramidines are also present in

A. Theophylline

B. Theobromine

C. Flavin mononucleotide

D. Thiamine

Purine ring is present in

Pyramidine ring is present in

Theobromine Thiamine

Theophylline

Flavin Mononucleotide

3. Which one of the following procedures as routine technique for karyotyping using light microscopy:

A C-banding

B G-banding

C Q-banding

D Brd V-standing

Ans. B) G-Banding

a. “The most commonly employed staining method uses a Giemsa stain and hence is called G banding”

b. A Karyotype is a standard arrangement of a photographed or image stained chromosomes, where chromosomes are in metaphase stage.

c. Mitosis is arrested in dividing cells in metaphase stage by use of colchicine.

d. In metaphase stage individual chromosomes take the form of two chromatids connected at the centromeres.

i. Chromosomes are arranged in pairs

ii. Chromosome pairs arranged in decreasing order of length.

Staining allows identification of each individuals chromosome on the basis of distinctive and reliable pattern of alternating light and dark bonds. One of the following banding technique may be used.

STAINING OF CHROMOSOME

G—Banding Q— Banding C-Banding R-Banding

Giemsa bonding

Quinacrine- banding

Constitutive-banding

Reverse stainingGiemsa banding

Most commonly used

Demonstrates bandsalong chromosome

Demonstrate constitutivehetero chromatin

Gives pattern opposite to G-banding

4. Which of the following is not a nitrogenous base

A. Adenine

B. Guanosine

C. Cytosine

D. Thymine

B. Guanosine

-Guanosine is a nucleoside

-Remaining are nitrogenous bases

-Guanine is a nitrogen base

5. On complete hydrolysis of DNA we will get all the following except

A Deoxy pentose sugar

B Phosphoric acid

C Adenosine

D Purine bases

C) Adenosine- Nucleoside = Deoxy - Adenosine or adenosine = Base (Adenine) + sugar

- Adenosine = ribose + Adenine

-Deoxyadenosine = Deoxyribose + Adenine

Therefore Adenosine is present in RNA, not present DNA.

-DNA is a Polymer of deoxyribonucleotides

i. Adenine deoxyribonucleotideii. Thymine deoxyribonucleotideiii. Guanine deoxyribonucleotideiv. Cytosine deoxyribonucleotide

- Nucleotides joint by covalent 3’-5’ phosphodiester linkage

-Right handed Double helix structure of DNA is given by Waston and Crick

-A combine with T (A = T) by two H2 bond C combines with G(C =G) by three H2 bond

-Chargaff’s Rule — Ratio of purine (G+A) to pyrimidine (T+C) bases in the DNA is always around 1. - i.e. G + A / T + C = 1

-Antiparallel i.e one strand in 5’-3’ direction and other in 3’-5’ direction

DNA - 6 types of DNA : A to E and Z

- most common TYPE of DNA is B-DNA

B-DNA-10 base pair per turn

-Most stable

-This was actually explained by watson and crick

-Length of one turn = 34 A* or 3.4nm

-Diameter oe width = 20 A* or 20nm

-Right handed helix ( Z-DNA is left handed)

DNA STRANDSTEMPLATE STRAND CODING STRAND

Non coding strand

Antisense strand Sense strand

-ve strand +ve strand

3’-5’ direction 5’-3’ direction

This strand is copied during m-RNA synthesis

6. Thermostability of DNA is provided by

A G-C bonds

B A-T paining

C N-glycosolic bond

D Antiparallel arrangement

Ans. A G-C Bonds

- ‘A’ combines with ‘T’ by 2 hydrogen bonds i.e A = T while ‘C’ combines with ‘G’ by 3-Hydrogen bondsi.e. C = G

Since it has 3 bonds it is responsible for its stability

Melting (Denaturation) of DNA

- Separation of 2 strands of DNA by breaking of Hydrogen Bonds

- DNA separated into two components strands either by:

1) increasing temperature or 2) decreasing salt concentration.

- Phosphodiester bond is not broken

- Primary structure is not disrupted, only secondary and tertiary structure is disrupted.

- After denaturation (melting), of DNA, there is an increase in the optical absorbance of purine and pyrimidine bases à a phenomenon referred as Hyperchromicity of denaturation.

- DNA rich in G=C melt at higher temperature than that rich in A=T pairs.

- Formamide is used commonly in recombinant DNA experiments, Lowers the ‘Tm’ by destabilizing H bonds.

Melting (Denaturation) of DNA-Melting Temperature (Tm) = Temperature at which half of strand is denatured

-Normal Tm = 85-95*C

-Tm = 2*no. of A=T Pairs + 3 * no. of C=G pairs

TRIPLEX DNA-3rd strand binds at major groove of B-DNA by Hydrogen bonding

-k/a Hoogsten Pairs

4-STRANDED DNA-High in Guanine content

- G –Quarlet DNA

TOPOISOMERASE- Regulates overwinding or underwining of DNA.

Topoisomerase I Topoisomerase II

Makes Single Stranded Nick In DNA

Makes double stranded Nick

Removes –ve supercoils Removes –ve supercoils

Cant insert supercoils Can insert supercoils

ATP is not requires ATP is required

E.g.: Helicase DNA gyrase

TOPOISOMERASE

DRUGS ACTING ON TOPOISOMERASEBacterial Topoisomerase

Human topoisomerase

Ciprofloxacin Etoposide

Nalidixic acid Adriamycin

Daunorubocin

NUCLEOSOME-DNA + Histone Protein

-Most abundant chromatin protein = Histone protein

-5 Types :

H1 Lysine rich

Linker proteinH2A, H2B

Arginine rich

H3 Lysine richH4

-Beads of String appearance

-Nucleosomes are linked to each other by 30bp k/a Linker.

- Basic protein

NUCLEOSOME

REGIONS IN DNAEUCHROMATIN HETEROCHROMATIN

Regions in DNA which is transcriptionally active.

transcriptionally inactive

Chromatin is Less densely packed Densely packed

Stains less densely Stains densely

TYPES OF HETEROCHROMATIN

Constitutive Facultative Always condensed At times condensed but other times

it is actively transcribed and thus uncondensed and appears as euchromatin

-Inactive-Seen in centromere and ends of telomere

X- chromosome

One X CHROMOSOME is inactive in females (barr body) but at time of embryogenesis it becomes active

-Y chromosome is Acrocentric

-X chromosome is submetacentric

-Telocentric chromosome is not seen in humans

-MC chromosome = Submetacentric

HYPERCHROMATISM-It is increase of absorbance

-Measured by absorbance at 260nm

-It occurs when DNA is denatured

-ssDNA is more Hyperchromatic than dsDNA.

DNA REPLICATION

DNA REPLICATION-Occurs in S phase of cell cycle

-Each DNA strands separates and acts as template strand on which complementary strand is synthesised

-Base pairing rule is obeyed

-Semiconservative nature

-New strand is synthesised in 5’-3’ direction

-Synthesis of DNA in both strands is not similar :

- Leading strand : DNA is continuosly polymerised

- Lagging strand : DNA is discontinuosly polymerised (semi discontinuous)

DNA REPLICATION-Replication proceeds from multiple origin in each chromosomes in eukaryotes including humans

-Replication obeys polarity

-Replication occurs in both directions along all of chromosome

-Both strands are replicated simultaneously

-Replication process generates ‘replication bubbles’

Common features between prokaryotic and eukaryotic DNA replication:

1) Semiconservative

2) Bidirectional

3) Semi discontinuous

DNA POLYMERASEIN EUKARYOTES

TYPES FUNCTION

DNA Polymerase Alpha Primase

DNA Polymerae Beta DNA repair

DNAP gamma Mitochondrial DNA synthesis

DNAP delta Lagging strand synthesis

DNAP epsilon Leading strand synthesis

DNA POLYMERASEIN PROKARYOTES

Polymerase I Gap filling following DNA replication, repair and recombination

Polymerase II Proof reading and repair

Polymerase III Leading strand synthesis, Okazaki fragment synthesis

PROTEINS INVOLVEDIN DNA REPLICATION

PROTEINS FUNCTIONDNA polymerases Deoxynucleotide polymerizationHelicases Unwinding of DNATopoisomerase Relieves torsional strain that

results from helicase induced unwinding

DNA Primase Initiates synthesis of RNA primers

Single stranded binding proteins

Prevents premature reannealing of dsDNA

DNA ligase Seals the broken ends between nascent chain and okazaki fragments

TELOMERASE-Telomeres are present at the end of eukaryotic chromosome

-Telomeres consist of TG repeats

-During each replication telomere shortens and thus cell dies later

-Early shortening of telomere is associated with early aging and malignancy

-Telomerase lengthens telomere and it is RNA dependent DNA polymerase(Reverse Transcriptase)

Q. Highly repetitive DNA is seen in (PGI)

A. Cloning of DNA

B. Microsatellite DNA

C. Telomere

D. Centromere

Ans. C. Telomere

D. Centromere

-In human DNA around 30% of genome consists of repeatitive sequence

-The sequence are clustered in centromere and telomere

-They are transcriptionally inactive

-They mostly have structural role in chromosome.

An enzyme called Helicase breaks the hydrogen bonds between the bases of the two antiparallel strands.

The strands are initially split apart in areas that are rich in A-T base pairs forming a replication fork.

DNA Gyrase (also called Topoisomerase) relieves tension that builds up as a result of unwinding.

Single strand binding proteins (SSBs) help to stabilise the single stranded DNA.

STEP-1

RNA polymerase (also known as RNA Primase) synthesizes short RNA nucleotides sequences that act as primers (starters).

These essentially provide a starting point for DNA replication.

STEP-2

STEP-3DNA Polymerase III can now start synthesising the new DNA strand using free DNA nucleotides.

However, DNA polymerase can only read the original template (parent strand) in the 3’ → 5’ direction (making DNA 5’ → 3’).

This is not a problem on the leading strand, because the DNA polymerase can simply continue to read along as the two parent stands continue to unzip.

STEP-4On the lagging strand DNA polymerase moves away from the replication fork.

As the strands continue to unzip more DNA is exposed and new RNA primers must be added.

As a result the lagging strand is synthesised in short bursts as DNA polymerase synthesizes DNA in-between each of the RNA primers.

STEP-5The newly synthesised lagging strand now consists of both RNA and DNA fragments.

The DNA fragments are known as Okazaki fragments, after a Japanese scientist who noticed that heating DNA during replication, which separates the strands, gave many small fragments of DNA.

From this he concluded that one stand must be synthesized in short bursts of DNA.

STEP-6DNA Polymerase I now removes the RNA primers and replaces them with DNA

STEP-7DNA Ligase joins the DNA fragments of the lagging strand together to form one continuous length of DNA.

DNA REPAIRDNA damaging agents

Defects in DNA

Repair mechanism

Disorder associated

UV lightsChemicals

Pyrimidine dimers

Nucleotide excision repair

Xeroderma pigmentosa

Replication errors

Mismatch repair

Ionising radiation

Homologous recombination

Ataxia telangiectasia

MOLECULAR BIOCHEMISTRY- II

TRANSCRIPTION

Key points:

Transcription is the process in which a gene's DNA sequence is copied (transcribed) to make an RNA molecule.

RNA polymerase is the main transcription enzyme.

Transcription begins when RNA polymerase binds to a promoter sequence near the beginning of a gene (directly or through helper proteins).

RNA polymerase uses one of the DNA strands (the template strand) as a template to make a new, complementary RNA molecule.

Transcription ends in a process called termination. Termination depends on sequences in the RNA, which signal that the transcript is finished.

TRANSCRIPTION

(a)To initiate the transcription process, RNA polymerase, shown as a large green blob, binds to a promoter sequence shown in dark green on a double-stranded DNA molecule.

(b)Once bound, RNA polymerase and its associated proteins bend the DNA to separate the two strands.

(c) A DNA sequence downstream of the promoter region is labeled the termination site, and it indicates where the transcription process will end.

RNA POLYMERASERNA POLYMERASE DNA POLYMERASE

No Primer is needed Primer is needed

No proof reading activity

proof reading activity

TYPE OF RNA POLYMERASE

MAJOR PRODUCTS

RNA Polymerase I rRNA

RNA Polymerase II mRNA, miRNA, SnRNA

RNA Polymerase III tRNA, 5s rRNA

-In Prokaryotes there is only one type of RNA Polymerase

-Eukaryotes :3 Types

PROMOTERS OFTRANSCRIPTION

Bacterial Promoters

Eukaryotic promoters

1) TATA Box 10 bp upstream of start site of Transcription

1) Golberg Hogness Box

-Similar to TATA Box

-25-35 bp upstream

2) TGG Box 35 bp upstream 2) CAAT Box 70-80 bp upstream

PROMOTERS OFTRANSCRIPTION

-Usually Promoters are located upstream of start site of Transcription

- But Promoters for RNA Polymerase III is located Downstream

-Promoters lie on the coding strand of DNA but not on Template strand

(b) During the elongation phase, RNA polymerase adds nucleotides to a growing mRNA chain.

The nucleotides of the mRNA are represented here as pink T-shaped molecules; a curved red arrow indicates that they are added to the three-prime end of the growing mRNA transcript.

Blue arrows show that the DNA is wound back up at the left side and is unwound at the right side as RNA polymerase moves along the template strand from left to right, as indicated by a green arrow.STEP TO PG-MD/MS - DR.AKIF A.B

DNA STRANDSTEMPLATE STRAND CODING STRAND

Non coding strand

Antisense strand Sense strand

-ve strand +ve strand3’-5’ direction or 5’-3’ direction

5’-3’ direction or 3’-5’ direction

This strand is copied during m-RNA synthesis

(c) When RNA polymerase reaches the termination site on the template DNA strand, the mRNA transcript is separated from the DNA.

Here, the synthesized mRNA is shown as a separate molecule below the double-stranded DNA, and a red arrow above the double-stranded DNA shows the release of the RNA polymerase from the DNA.

POST TRANSCRIPTIONALPROCESSING

- Primarily occurs at nucleus

1) 7-methylguanosine capping at 5’ end

3) Removal of Introns and Joining of Exons called splicing

2) Addition of Poly A tail at 3’ end

RNARNA DNA

Mostly seen in cytoplasm Nucleus

Destroyed by alkali Not destroyed

Single stranded Double stranded

NON CODING RNAs1)Transfer RNA (tRNA)

2)Ribosomal RNA (rRNA)

3) mi RNA

4) Si RNA

RIBOSOMAL RNA-Most abundant RNA

-40S Ribosome = 18 SrRNA + 30 Proteins

-60S Ribosome = 5S rRNA + 5.8 + 28S

-Synthesised in Nucleolus

-Some rRNA acts as Ribozymes

tRNA-Transfer RNA

-Transfers Amino acids from Cytoplasm to Ribosomes

Acceptor arm -Amino acid binding site- CCA at 3’end

Anticodon arm Binds with mRNAD-ARM Dihydrouridine arm

- Detects aminoacyl tRNA Synthase

ARM - Ribothymidine and Pseudouridine

MODIFIED tRNA1) Dihydrouridine = One of double bond is

decreased

2) Pseudouridine : Ribose and N2 base is linked by C-C bond instead of C-N bond

3) Ribothymidine : methylation of one of uracil to form Thymine

- Only RNA to contain Thymine is : Ribothymidine tRNA

4) Inosine : Contain Hypoxanthine

1) Reverse Transcriptase : RNA dependent DNA Polymerase

2) DNA Polymerase : DNA dependent DNA Polymerase

3) Primase: DNA dependent RNA Polymerase

4) RNA Polymerase : DNA dependent RNA Polymerase

Replication Transcription

Deoxyribonucleotides are added

Ribonucleotides are added

Adenine pairs with Thymine Adenine pairs with Uracil

Both strands of DNA acts as Template

One strand act as template and other as Coding strand

A Primer is involved as DNA polymerase cannot initiate DNA synthesis on its own

Primer is not required

DNA dependent DNA Polymerase is the enzyme

DNA dependet RNA polymerase is the enzyme

TRANSLATION

CODON-Triplet nucleotide sequence present in mRNA representing specific Amino Acid.

-If 1 base represents 1 amino acid = then Total 4 Amino Acids (4 )

-If 2 base represents 1 amino acids = 4 = 16 amino acids

-If 3 base = 4 = 64 Amino Acids

-If 4 base = 4 = 256

TRANSLATIONEUKARYOTIC PROKARYOTIC

mRNA is monocistronic PolycistronicTranslation occurs only once transcription is complete

Translation can occur even before transcription is complete

Initiating Amino Acid = Methionione

N-Formyl Methionine

- mRNA is always translated from 5’ to 3’ direction.

STEP-1Charging of t-RNA

-Specific Amino acid is attached to acceptor arm(3’) of t-RNA by Amino-acyl-t-RNA synthase

-2 ATPs are used

-Amino acyl t-RNA synthase is specific for a Amino acid and t-RNA

-Responsible for high fidelity of Translation of Genetic message

There is no t-RNA for Hydroxylysine and Hydroxyproline since they are

formed by post translation modification of Lysine and Proline

STEP-2INITIATION

STEP 2ARIBOSOMAL DISSOCIATION

80S Ribosome = 60S + 40 S

-2 sites for t-RNA on Ribosomes = P site( Peptidyl) + A site (Amino acyl)

-Initially t-RNA bind to P-site

STEP-2BEIF2 + GTP

EIF2 - GTP

EIF2 –GTP-t-RNA

43 S preinitiation

complex Binds to AUG on m-RNA at 5’

48S initiation complex

Binds with 60S

ribosomes

Forms 80S initiation complex

Release of all

elongation factors(Eif2-

Methionine-t-RNA

Binds to 40S ribosomes

T-RNA is at P site

SHINE –DALGARNO SEQUENCE

-In Prokaryotes

- Codon sequence near initiator codon which facilitates binding of Pre-Initiator complex with m-RNA

KOZAK-COSENSUS SEQUENCE

-In Eukaryotes

- Codon sequence near initiator codon which facilitates binding of Pre-Initiator complex with m-RNA

STEP-3ELONGATION PHASE

STEP-3A

STEP-3B-New amino acid binds with t-RNA and then this forms complex with GTP to attach to A site of Ribosome on m-RNA

STEP-3 C,D,E

STEP-3F

STEP-3G

STEP-4TERMINATION

There are three termination codons that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA.

No tRNAs recognize these codons.

Thus, in the place of these tRNAs, one of several proteins, called release factors, binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.

INITIATOR CODONS

-In Eukaryotes= AUG codes for Methionine

-In prokaryotes = AUG codes for N-Formyl-Methionine

ENERGY REQUIREMENTSDURING TRANSLATION

Charging of t-RNA 2 Phosphates

Formation of 48 S Pre initiation complex

Formation of 80 S initiation complex

Binding of fresh Aminoacyl t-RNA @ A- SITE

During Translocation 1 GTP

During Termination 1 GTP

TERMINATOR CODONSUAG UGA UAA

Amber Opal Ochre

EXCEPTIONSUAG can be recoded to

PyrollysineUGA can be recoded to

Selenocysteine

UGA also codes for Tryptophan in Mitochondrial DNA

POST-TRANSLATIONALMODIFICATIONS

1) Trimming2) Glycosylation -N-Glycosylation occurs in Endoplasmic

Reticulum

-O-Glycosylation occurs in Golgi Apparatus

3) Hydroxylation Of proline and Lysine gives Hydroxyproline and Hydroxy Lysine

4) Gamma Carboxylation

Of Vit. K dependent clotting factors ( 2,7,9,10)

5) Protein folding Chaperons6) Protein degradation

UbiquitinSTEP TO PG-MD/MS - DR.AKIF A.B

Molecular biochemistry

Health & Medicine

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