DNA, RNA and Protein Synthesis

Objective:

To construct using three dimensional models or diagrams the structure and role of a “gene” in protein synthesis, including the concept of gene expression, promoter sequence, exons, and introns, at the level of 85% proficiency for each student.

 

In order to achieve this objective, you will need to be able to:

 

  1. Examine and identify the structure of nucleotides, nucleic acids, RNA and DNA.
  2. Define cDNA vs DNA, precursor-RNA, messenger-RNA, transfer-RNA, ribosomal-RNA, codon and anti-codon.
  3. Describe the role of DNA and RNA in the process of protein synthesis.
  4. Explain the significance of the organization of a "gene", including the regions for the Promoter, the Signal Protein, the Material Protein, and the Exons and Introns.

Materials:

Group Supplies

Computer with the Chime plug-in installed for Internet Explorer

http://www.mdlchime.com

(The Chime plug-in has been pre-installed onto the computers in the physiology lab)

 

Molecular structure library from the ChemIDplus System at the National Library of Medicine

http://chem.sis.nlm.nih.gov/chemidplus/setupenv.html

(Selected files have been downloaded and pre-loaded onto the computers in the physiology lab)

 

Structures of DNA, Nucleic Acids and Codons

http://molvis.sdsc.edu/dna/index.htm

(These files have been downloaded and pre-loaded onto the computers in the physiology lab)

 

The nucleotide sequence of the human “AVP” gene

http://www.gdb.org ; http://www.ncbi.nlm.nih.gov

 

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=Search&term=M11166&doptcmdl=GenBank

 

Methods and Results:

Double click the shortcut named 3d_dna to select three dimensional reconstructions of nucleotides and DNA:

 

 

Nucleotide structure

The students at each lab table will draw a different nucleotide

Lab table 1

Adenine Nucleotide

Lab table 2

Thymine Nucleotide

Lab table 3

Cytosine Nucleotide

Lab table 4

Guanine Nucleotide

Use three dimensional computer images to draw models of Deoxyribose and a Phosphate Group.  Covalently bond the Deoxyribose to the Phosphate group.  Make certain that the Phosphate group attaches to carbon # 5 of the Deoxyribose.

Use the three dimensional computer images to draw the nucleotide base assigned to your table.  Covalently bond your nucleotide base to the deoxyribose.  Make certain that the correct Nitrogen of the base attaches to carbon # 1 of the Deoxyribose.

DNA structure – single strand

All tables will work together to draw a short single strand of DNA.  Confirm that the phosphate of your first nucleotide will covalently bond to carbon # 3 in the Deoxyribose of the next nucleotide.

DNA duplication

Because nucleotides are fairly complex, shorthand expressions are often used. Take the nucleotide sequence of the following single strand of DNA:

T  A  C  G  G  A  C  T  G

DNA nucleotide sequence

Generate the sequence of the complementary strand of DNA.  This is called complimentary DNA (cDNA)

 

cDNA nucleotide sequence

To produce a double strand of DNA, join the two single strands.

T  A  C  G  G  A  C  T  G

DNA nucleotide sequence

 

cDNA nucleotide sequence

Separate the double strands and make complementary strands of each

T  A  C  G  G  A  C  T  G

DNA nucleotide sequence

 

cDNA nucleotide sequence

 

 

DNA nucleotide sequence

A  T  G  C  C  T  G  A  C

cDNA nucleotide sequence

DNA transcription

Take the nucleotide sequence of the following strand of DNA

T  A  C  G  G  A  C  T  G

DNA nucleotide sequence

Generate the sequence of the complementary strand of RNA

 

pRNA nucleotide sequence

(The pRNA can duplicate itself and produce a double strand).  The pRNA may leave the nucleus as mRNA and be used to synthesize protein.

Protein Synthesis

The mRNA attaches to ribosomes to orchestrate the attachment of the tRNA.  Each three nucleotides of mRNA will attach in sequence to tRNA that contain complementary sequences of three nucleotides.  Groups of three nucleotides in mRNA are referred to as Codons.  The groups of three nucleotides in the tRNA are referred to as Anticodons.  A particular codon will represent a particular amino acid attached to the tRNA.

Take the nucleotide sequence of the following strand of mRNA

A  U  G  C  C  U  G  A  C

mRNA nucleotide sequence

Generate the sequence of tRNA nucleotides that will complement and attach to the mRNA

 

tRNA nucleotide sequence

Determine the amino acids that will be brought together by the tRNAs.  The coding for the amino acids based on mRNA is shown on the next page.

A

U

G

C

C

U

G

A

C

 

 

 

 

 

The Genetic Code (mRNA)

 

 

The Genetic Code (mRNA)

1st position
(5' end)

2nd position (middle)

3rd position
(3' end)

U

C

A

G

U

Phe F
Phe F
Leu L
Leu L

Ser S
Ser S
Ser S
Ser S

Tyr Y
Tyr Y
STOP
STOP

Cys C
Cys C
STOP
Trp W

U
C
A
G

C

Leu L
Leu L
Leu L
Leu L

Pro P
Pro P
Pro P
Pro P

His H
His H
Gln Q
Gln Q

Arg R
Arg R
Arg R
Arg R

U
C
A
G

A

Ile I
Ile I
Ile I
Met M

Thr T
Thr T
Thr T
Thr T

Asn N
Asn N
Lys K
Lys K

Ser S
Ser S
Arg R
Arg R

U
C
A
G

G

Val V
Val V
Val V
Val V

Ala A
Ala A
Ala A
Ala A

Asp D
Asp D
Glu E
Glu E

Gly G
Gly G
Gly G
Gly G

U
C
A
G


Decoding the “AVP” gene

The human “AVP” gene contains 2500 base pairs.  Within these base pairs are:

Find each of the sequences above using the cDNA nucleotide sequence of the human “AVP” gene.  (Reproduced on the following pages)

Take the cDNA nucleotide sequence and construct the amino acid sequence for 8-arginine vasopressin

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

297

298

299

300

301

302

303

304

305

306

307

308

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

From the National Library of Medicine

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=Search&term=M11166&doptcmdl=GenBank

 

M11166. Human prepro-8-ar...[gi:340302]

 

LOCUS       HUMVPNP                 2500 bp    DNA     linear   PRI 02-MAY-1996

DEFINITION  Human prepro-8-arginine-vasopressin-neurophysin II gene, complete

            cds.

ACCESSION   M11166

VERSION     M11166.1  GI:340302

KEYWORDS    neurophysin; vasopressin.

SOURCE      Homo sapiens (human)

  ORGANISM  Homo sapiens

            Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;

            Mammalia; Eutheria; Primates; Catarrhini; Hominidae; Homo.

REFERENCE   1  (bases 1 to 2500)

  AUTHORS   Sausville,E., Carney,D. and Battey,J.

  TITLE     The human vasopressin gene is linked to the oxytocin gene and is

            selectively expressed in a cultured lung cancer cell line

  JOURNAL   J. Biol. Chem. 260 (18), 10236-10241 (1985)

  MEDLINE   85261445

   PUBMED   2991279

COMMENT     Original source text: Homo sapiens (clone: hAVP4.) placenta DNA.

            A draft entry and printed copy of the sequence in [1] was kindly

            provided by J.Battey 07-FEB-1986.

FEATURES             Location/Qualifiers

     source          1..2500

                     /organism="Homo sapiens"

                     /mol_type="genomic DNA"

                     /db_xref="taxon:9606"

                     /map="20"

                     /clone="hAVP4."

                     /tissue_type="placenta"

     gene            175..2344

                     /gene="AVP"

     exon            175..344

                     /gene="AVP"

                     /note="G00-119-009"

                     /number=1

     CDS             join(225..344,1718..1919,2087..2259)

                     /gene="AVP"

                     /note="precursor"

                     /codon_start=1

                     /product="vasopressin-neurophysin II"

                     /protein_id="AAA98772.1"

                     /db_xref="GI:340303"

                     /db_xref="GDB:G00-119-009"

                     /translation="MPDTMLPACFPGLLAFSSACYFQNCPRGGKRAMSDLELRQCLPC

                     GPGGKGRCFGPSICCADELGCFVGTAEALRCQEENYLPSPCQSGQKACGSGGRCAAFG

                     VCCNDESCVTEPECREGFHRRARASDRSNATQLDGPAGALLLRLVQLAGAPEPFEPAQ

                     PDAY"

     sig_peptide     225..281

                     /gene="AVP"

                     /note="vasopressin-neurophysin II signal peptide"

     mat_peptide     282..308

                     /gene="AVP"

                     /product="8-arginine-vasopressin"

                     /note="8-arginine-vasopressin; G00-119-009"

     mat_peptide     join(318..344,1718..1919,2087..2136)

                     /gene="AVP"

                     /product="neurophysin II"

                     /note="G00-119-009"

     intron          345..1717

                     /gene="AVP"

                     /note="G00-119-009"

                     /number=1

     exon            1718..1919

                     /gene="AVP"

                     /note="G00-119-009"

                     /number=2

     intron          1920..2086

                     /gene="AVP"

                     /note="G00-119-009"

                     /number=2

     exon            2087..2344

                     /gene="AVP"

                     /note="G00-119-009"

                     /number=3

ORIGIN      At Sau3A site.

 

        1 gatcccctgc acagacaggc ccacgtgtgt ccccagatgc ctgaatcact gctgaccgct

       61 ggggacctgg cggccgtggg ctcctgggga gccactgggg agggggtggc ggccgcgtct

      121 cgcctccacg ggaacacctg cggacataaa taggcagcca gcagaggcag cagcacagag

      181 ccaccaagca gtgctgcata cggggtccac ctgtgtgcac caggatgcct gacaccatgc

      241 tgcccgcctg cttccctggc ctactggcct tctcctccgc gtgctacttc cagaactgcc

      301 cgaggggcgg caagagggcc atgtccgacc tggagctgag acaggtactt cccactgtgg

      361 gccatctcag ggcagccata gcgggcagtg ctgacaccct gggtcagggg ctaggaaaga

      421 gggaagtcat gggtggtggt agcctttagg ggaagttcgg gggaggaaga gggaggcatg

      481 gcatggctgg gcagaggagc caatggggtg ggccagaggg gaccaggctt tggaggaggc

      541 tgggagaggc tgaaggcgct cctggtcact gtcgccatcc agacagggat gcaggaaaat

      601 gagggatgct tccccggtga ctgggcttgg ggctggatag ggagaacggg gcatcatggc

      661 ctcccctgtg cccatggcgt tcttgcatct ggactggctg gggcagcaga ggctccatcc

      721 tacctagcat tggaggcttt cctcatccag ccccagcctc ccagccacag gcgcccaggc

      781 ccccacacag aagatggcca ctggtctgag cgcgcttgag tggggcatcc tgtgggaagt

      841 tctgctggga acctggccta attctatagt gctggacgtt tcctccattt ccagcagagc

      901 tgaaggaaat ccaatcacga tgtgcatgca attctgtcca gctcaatgat gagcccttga

      961 gcaaattaga ccacaccagg ctcacgtaaa agtctaatcg cgtatccatt gcgccagaga

     1021 accggctgtt gagcagatga gagtgcgcgc tcggcaaccc ccgcagcctc tcttcctcct

     1081 gctaggctcc tttagggtcc tgagggcacc tgggtgtccg tgctcgcctc taggtctcag

     1141 cccctgccac ccacctgata ggtcataggt ggctgagcag gggtcagggc tccagctgag

     1201 gccgacaagc ttggcggggc cagggcgaag gcaagagagg agacaggaaa tgggaagggc

     1261 cggggttctg gatgggtagg gcctctccgc atggtgtagt ggggaagggg gtgggcccgg

     1321 gctcaagccg cagcagggcg aggaggaagg aggaagggtc tggagtggtg gagggtgggg

     1381 cagctgcaac agtggcgccc accagcgatg accccgaggc tcgaggaagg gctccccacg

     1441 ctgtagtcca cgggagaccc gaccctagct gagggtgagg acgctgaggg ctgtcaccga

     1501 gaggtcatcc aagaaaccaa ggtgccgagc agatctggac gccccgcccg tgaccgcggt

     1561 cgaggcccag tggcgcccga gcgtgcctgc agccgcagcc ccggtgtccc gcccgcactc

     1621 cgagccctgg accccagcat ccccgcctcg ctgcgttccc ctccaacccc tcgactcccg

     1681 gctcccctcc tcccgctcac cccgcccgtc cccgcagtgc ctcccctgcg gccccggggg

     1741 caaaggccgc tgcttcgggc ccagcatctg ctgcgcggac gagctgggct gcttcgtggg

     1801 cacggctgag gcgctgcgct gccaggagga gaactacctg ccgtcgccct gccagtccgg

     1861 ccagaaggcg tgcgggagcg ggggccgctg cgccgccttc ggcgtttgct gcaacgacgg

     1921 tgcgcggcgg gggcgggcct gggggggggc gcagacgctt gggtgggggg gacgcgggcc

     1981 tgcggcgggg tgggggctcg tgcgggcccg gcagggaggg tgtgggcccc ccgcaccccg

     2041 agctgcgccc gccccaggcg cccgtgctca cacgtcctcc cggcagagag ctgcgtgacc

     2101 gagcccgagt gccgcgaggg ctttcaccgc cgcgcccgcg ccagcgaccg gagcaacgcc

     2161 acgcagctgg acgggccggc cggggccttg ctgctgcggc tggtgcagct ggccggggcg

     2221 cccgagccct tcgagcccgc ccagcccgac gcctactgag cccgcgctgc ccccaccgcg

     2281 ctcttccgcc cgcccctgca gcacggacaa taaacctccg ccaatgcacg gcctcgcgtc

     2341 tgtctcagtc tctggcggga agagggaagg ggagagaggt gggagcgcgg acccccgcca

     2401 ccacgcccac cggccagtcc ccggacctga ggtcgtgggc agatccaccc cagagaagca

     2461 acaggtcccg tagaggaagc gatctgggac ccgcagaggt

//

position 1-168

A Promoter sequence that binds to RNA polymerase and determines which strand of DNA is transcribed

position 169-224

A code for controlling sequences

position 225-281

A Signal protein code that codes for a protein that directs the material protein to specific cellular organelles

position 282-308

The code for 8-Arginine Vasopressin that codes for vasopressin – antidiuretic hormone

positions 318-344; 1718-1919; 2087-2136

The code for Neurophysin II that codes for a protein that transports vasopressin in blood

position 2138-2343

The code for a Glycoprotein that codes for a glycoprotein

position 345-1717

The code for Intron 1

position 1920-2086

The code for Intron 2

position 2344-2500

The terminator code

 

Discussion:

  1. Compare and contrast the structure of RNA and DNA
  2. Explain the role of the promoter region of a gene in gene expression?
  3. Speculate on the role of chemical messengers in the control of gene expression.
  4. Speculate on the significance of the introns in genetic expression..

 

© David G. Ward, Ph.D.  Last modified by wardd 23 May, 2006