chapter 25 nucleic acids and protein synthesis 25.1 introduction the nucleic acids, deoxyribonucleic...
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CHAPTER 25
NUCLEIC ACIDS AND PROTEIN SYNTHESIS
25.1 INTRODUCTION
The nucleic acids, deoxyribonucleic (DNA) and ribonucleic (RNA) , are, respectively , the molecules that preserve here-ditary information and that transcribe and translate it in a way that allows the synthesis of all the varied proteins of the cell
25.2 NUCLEOTIDES AND NUCLEOSIDES
Mild degradations of nucleic acids yield their monomeric units, compounds that are called nucleotides.
OPOH2C
OH OH
N
O
HO
HO
1'
2'3'4'
5'
Heterocyclic base
-N-Glycosidic linkage
O
POH2C
OH OH
O
HO
HO
1'
2'3'4'
5'N
N
N
N
NH2
1
2
3
4
567
8
9
Adenylic acid ( ÏÙÜÕËá)
A nucleotide (ºËÜÕ)
Complete hydrolysis of a nucleotide furnishes:1. A heterocyclic base, either a purine or pyrimidine.2. a five-carbon monosaccharide, either D-ribose or 2-deoxy- D-ribose.3. A phosphate ion.
The central portion of the nucleotide is the monosaccharide and it is always present as a five-membered ring, that is, as a furanoside.
The nucleosides that can be obtained from DNA all contain 2-deoxy- D-ribose as their sugar component and one of four heterocyclic bases,either adenine, guanine, cytosine, or thymine.
N
N
N
N
NH2
H
N
N
N
N
NH
H
H
NH2 N N
NH2 O
O O
H
HH3C
H
Adenine(ÏÙàÑßÊ)
Guanine(ÄñàÑßÊ)
Cytosine(°ûºËà×à¤)
Thymine(ÐØÏÙà×à¤)
Purines Pyrimidines
The nucleosides obtained from RNA contain D-ribose as their sugarcomponent and either adenine, guanine, cytosine, or uracil as theirheterocyclic base.
N
N
O
O
H
H
Uracil(Äòà×à¤)
Nucleotides are named in several ways. Adenylic acid, for example,is sometimes called 5’-adenylic acid, it is also called 5’-phosphate,or simply adenosine monophosphate(AMP).
The names and structures of the nucleoside found in DNA and RNA:
N
N
N
N
NH2O
O O
HH3C
CytosineThymine
OCH2OH
OH H
HH H
H
OCH2OH
OH H
HH H
H
2'-Deoxycytidine 2'-Deoxythymidine
AdenineGuanine
OCH2OH
OH H
N
N
N
N
NH2
HH H
H
OCH2OH
OH H
N
N
N
N
O
HH H
H
H
NH2
2'-Deoxyadenosine (2'-ÍÑÑõÏÙÜÕ)
2'-Deoxyguanosine (2'-ÍÑÑõÎ »ÆÜÕ)
N
N
N
N
NH2O
O O
HH3C
AdenineGuanine
CytosineThymine
OCH2OH
OH OH
N
N
N
N
NH2
HH H
H
OCH2OH
OH OH
N
N
N
N
O
HH H
H
H
NH2
OCH2OH
OH OH
HH H
H
OCH2OH
OH OH
HH H
H
Adenosine (ÏÙÜÕ)
Guanosine (ÄñÜÕ)
Cytidine (°ûà¤)
Uridine (ÄòÜÕ)
25.3 LABORATORY SYNTHESIS OF NUCLEOSIDES AND NUCLEOTIDES
One technique uses reaction that assemble the nucleoside from suitably activated and protected ribose derivatives and heterocyclic base.
N
N
N
N
NHCOCH3
HgCl
OCH2OCOCH3
OCOCH3OCOCH3
H
Cl+ -HgCl2 N
N
N
N
NHCOCH3
OCH2OCOCH3
OCOCH3OCOCH3
OH-
H2O Adenosine
Another technique involves formation of the heterocyclic base on a protected ribosylamine derivative.
OC6H5OCOH2C
OCOC6H5OCOC6H5
NH2
+OH-
H2O UridineHC
CH
OC2H5
CNH
O
C O
OC2H5
(-2C2H5OH)
OC6H5OCOH2C
OCOC6H5OCOC6H5
N
N
O
O
H
The third technique involves the synthesis of a nucleosides with asubstituent in the heterocyclic ring that can be replaced with other groups.
OCH2OH
OH OH
N
N
N
N
Cl
HH H
H
NH3
H2S
Ni
N
N
N
N
NH2
R
N
N
N
N
S
R
H
N
N
N
N
R
R =
Specific phosphorylation of the 5’-OH can be achieved if the 2’- and 3’-OH groups of the nucleoside are protected by an isopropylidene group.
OCH2OH
O O
CH3H3C
base
+ (C6H5CH2O)2P_Cl
OPytidine
O(C6H5H2CO)2POH2C
O O
CH3H3C
base
O
(1) H3O+, H2O(2) H2 / Pd
OPOH2C
OH OH
base
O
HO
HO
25.3A MEDICAL APPLCATIONS
The discovering of the 6-mercaptopurine led to the development of other purine derivatives and related compounds. For example:
N
N N
N
H
SH
N
N N
N
H
OH
N
N N
N
O
O
HO
H
H2N
6-Mercaptopurine (6-ÛÏ(»ù)àÑßÊ)
Allopurinol (±ðàÑßÊ ¼)
Acyclovir
25.4 DEOXYRIBONUCLEIC ACID: DNA
25.4A PRIMARY STRUCTURE
Nucleotides bear the same relation to a nucleic acid that amino acids do to a protein; they are its monomeric units.
N
N
N
NH2
O
O
O
HH3C
OCH2
O
OCH2
O
OCH2
O
N
N
N
N
NH2
OCH2
O
N
N
N
N
O
H
NH2
P OO
O-
P OO
O-
P OO
O-
5' end
3' endP OO
O-
3'
3'
3'
3'
5'
5'
5'
5'
Phosphate ester linkathe 3’-OH of one ribosewith the 5’-OH of anther
The connection liksin protein are aminogroups; in nucleic acidsthey are phosphate esterlinkages.
5' 3'A T G C
25.4B SECONDARY STRUCTURE
The secondary structure of DNA is important because it enables us to understand how the genetic information is preserved, hoe it canbe passed on during the process of cell division, and how it can be transcribed to provide a template for protein synthesis.
Chargaff pointed out that for all species examined:
1. The total mole percentage of purines is approximately equal to that of the pyrimidines, that is, (%G +%A) / (%C + %T) ≈12. The mole percentage of adenine is nearly equal to that of thymine (i.e.. %A / %T≈1) and the mole percentage of guanine is nearly equal to that of cytosine (i.e.. %G / %C≈1)3. The ratio that varies from species is the ratio (%A + %T) / (%G + %C).
Watson and Crick had X-ray that gave them the bond lengths and angles of the purines and pyrimidines of model compounds. They also observed that the internal distance of the double helix is such that it allows only a purine-pyrimidine. Type of hydrogen bondingbetween base pairs.
Base pairing through hydrogen bonds can in only a specific way:
N
N
H3C O
O
N
N
N
NN
H
H
H¡ ¡
¡ ¡
N
N
N
O
N N
NO¡ ¡
¡ ¡
H
H
NH
H
H¡ ¡
Thymine(ÐØÏÙà×à¤)
Adenine(ÏÙàÑßÊ)
Cytosine(°ûºËà×à¤)
Guanine (ÄñàÑßÊ)
Specific base pairing of this kind is consistent with Chargaff’s finding that the %A / %T≈1 and that the %G / %C≈1.
The two chains of DNA are complementary. Where adenine appears in one chain, thymine must appear opposite it in the other; wherevercytosine appears in one chain, guanine must appears in the other. A always pairs with T and G always pairs with C.
25.4C REPLICATION OF DNA
Just prior to cell division the double strand of DNA begins at one end.complementary strands are formed along each chain.
(a) There are approximately 6 billion base pairs in the DNA of a single human cell.(b) The weight of DNA in a single human cell is 6¡Á10-12g
25.5 RNA AND PROTEIN SYNTHESIS
Protein synthesis requires that two major process take place; the first takes place in the cell nucleus, the second in the cytoplasm. The first is transcription, a process in which the genetic messenger is trans-cribed on to a form of RNA, called messenger RNA(mRNA). The second pro-cess involves two other forms of RNA, called ribosomal RNA(rRNA) and Transfer RNA (tRNA).
25.5A MESSENGER RNA SYNTHESIS—TRANSCRIPTION
Protein synthesis begins in the cell nucleus with the synthesis of mRNA. The ribonucleotide units of mRNA are joined into a chain by an enzyme called RNA polymerase then the chain separate into mRNA.
Then the mRNA migrates into the cytoplasm where it acts as a template for protein synthesis.
25.5B RIBOSOMES—rRNA
Scattered throughout the cytoplasm of most cells are small bodies called ribosomes. Although the ribosomes are at the site of protein synthesis, rRNA itself does not direct protein synthesis. Instead, a number of ribosomes become attached to a chain of mRNA and form what is called a polysome. One of the function of rRNA is to bind the ribosome to the mRNA chain.
25.5C TRANSFER RNA
Transfer RNA has a much low molecular weight and much moresoluble than mRNA or rRNA. The function of tRNA is to transportamino acids to specific areas of the mRNA of the polysome.
25.5D THE GENETIC CODE
Genetic code: triplet on mRNA corresponds to amino acid.
The code must be in the form of three bases, because there are 20 different amino acids used in protein synthesis but there are only four different bases in mRNA.
Lets see the synthesis of a hypothetical polypeptide:
A tRNA bearing Metformyl uses its anticodon to associate with the proper codon(AUG) on that portion of mRNA that is in contact with a ribosome. The next triplet of bases on this particular mRNA chain is AAA; this is the codon that specifies lysine. A lysyl-tRNAwith the matching anticodon UUU attaches itself to this site. The two amino are in the proper position for an enzyme to join themin peptide linkage. Then the ribosome moves down in contract
with the next codon. This one, GUA, specifies valine. A tRNA bearing valine binds itself to this site. Another enzymatic reactions takes place attaching valine to the polypeptide chain.Then the whole process repeats itself again and again. Even beforethe polypeptide chain is fully grown, it begins to form its own specific secondary and tertiary structure.
25.6 DETERMINING THE BASE SEQUENCE OF DAN
The basic strategy of sequencing DNA includes cleaving them into manageable fragments, identifying points of overlap, and revealing the nucleotide sequence of the original nucleic Acid.
The first part of the process is accomplished by using enzymes called restriction endonucleases. These enzymes cleave double-stranded DNA at specific base sequences. Sequencing of the frag-ments can be done chemically or with the aid of enzymes.
25.6A CHEMICAL SEQUENCING
The double-stranded restriction fragment to be sequenced isenzymatically, separated , isolated and treated the labeled fragmentwith reagent. For example, if we had a chain like the following:
GCAATCACGTC32P
Treating the fragment with hydrazine. This will produce the following set of 5’-labeled fragment.
GCAATCACGT32P
GCAATCA32P
GCAAT32P
G32P
These fragment can then be separated by a technique called gelelectrophoresis.
The DNA to be sequenced may be cleaved next to specific pairs by subjecting it in separate aliquots to four different treatment. After cleavage, the separate aliquots are subjected to simultaneouselectrophoresis in four parallel tracks of the gel. After autoradio-graphy, the results allow reading of the DNA sequence directly from the gel.
25.7 LABORATORY SYNTHESIS OF OLIGONUCLEOTIDES
The traditional approach to genetics involves randomly altering or deleting genes by inducing mutations in an organism, and then observing the effects in its progeny. With high organism, the method is disadvantage.
The new approach-reverse genetics is to start with a cloned gene andto manipulate it in order to find out how it functions. One of the manipulation is to synthesize strands of DNA which called antisensenucleotides, are capable of binding with what is called the sense sequence of the DNA. In doing so, they can alter the activity of thegene, or even turn it off entirely. For example, if the sense portion of DNA in a gene read:
A—G—A—C—C—G—T—G—G
The antisense oligonucleotide would read:
T—C—T—G—G—C—A—C—C
Synthesis of such oligonucleotides is an active area of researchtoday and is directed at many viral diseases, including AIDS.
25.8 THE POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is an extraordinarily simpleand effective method for amplifying DNA sequences.
The PCR has already had a major effect on molecular biology. It is being used in medicine to diagnose infectious and genetic diseases.it is now being applied to the prenatal diagnosis of a number of genetic diseases, including muscular dystrophy, cystic fibrosis andso on.
The PCR has been used in forensic sciences, in human genetics, andin evolutionary biology.
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