5 DNA RNA Protein Synthesis

DNA, RNA, and Protein Synthesis

Importance and Structure of DNA: Deoxyribo-Nucleic Acid

Historical Review: 1900’s – Morgan’s studies with fruit flies

showed that genes were located on chromosomes and chromosomes consisted of protein and DNA

1952- Hershey-Chase demonstrated that DNA (not protein) was the genetic material of a viral phage

Figure 16.2a The Hershey-Chase experiment: phages

Figure 16.2b The Hershey-Chase experiment

Phages Infecting a bacterium

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Figure 16.1 Transformation of bacteria – Griffith (and later Avery, McCarty and MacLeod)

The Structure of DNA Nucleotide monomers:

Phosphate Pentose Sugar (C5) –

Deoxyribose Sugar Organic Nitrogen Base :

• Cytosine (C)• Adenine (A)• Guanine (G)• Thymine (T)

Structure of DNA cont’ Polynucleotide chain

with linkage via phosphates to next sugar, with nitrogen base away from backbone of

Phos-sugar-phos-sugar

Dehydration synthesis

Beginning of the 1950’s several labs Beginning of the 1950’s several labs were studying the structure of DNAwere studying the structure of DNA

Maurice Wilkins & Rosalind Franklin X-ray crystallography: x-rays pass

through pure DNA and diffraction of x-rays were then examined on film

James Watson and Francis Crick did not have the expertise of Franklin and were without proper photos until………

Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA

Watson and CrickFigure 16.0 Watson and Crick Figure 16.0x James Watson

April 1953 – Classical one page paper in Nature by Watson and Crick

A double helix – 2 polynucleotide strands Sugar-phosphate chains of each strand

are like the side ropes of a rope ladder Pairs of nitrogen bases, one from each

strand, form the rungs or steps The ladder forms a twist every 10 bases

(all from x-ray studies!)

Figure 16.5 The double helix

Internal Structure of DNA: Purine and pyridimine? REMEMBER X-RAY DATA

Confirms Erwin Chargaff’s Rules Confirms Erwin Chargaff’s Rules

# of Adenine = to # of thymine

# of guanine equal to # of cytosine

This dictates the combinations of N-bases that form steps/rungs

Does not restrict the sequence of bases along each DNA strand

Information storage in DNA The 4 nitrogenous bases are the

“alphabet” or code for all the traits the organism possesses

Different genes or traits vary the sequence and length of the bases

ATTTCGGAC vs. GGGATTCTAG vs. GATC

Replication/Duplication of DNA Due to complimentary base paring – one

strand of DNA determines the sequence of the other strand

Therefore, each strand of double stranded DNA acts as a template

The double helix first unwinds – controlled by enzymes –and uses new nucleotides that are free in the nucleus to copy a complimentary strand off the original DNA strand

Figure 16.7 A model for DNA replication: the basic concept (Layer 1)

Figure 16.8 Three alternative models of DNA replication

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1)

There are a series of enzymes that control DNA replication – enzymes which:

Uncoil the original double helix strand via a helicase

Single-strand binding protein keeps helix apart so replication can start

Prime an area to start replication – primase except it adds RNA nucleotides at first

Polymerase to join individual nucleotides (dehydration synthesis)

Ligases to join short segments

DNA REPLICATION

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Figure 16.10 Origins of replication in eukaryotes

Figure 16.11 Incorporation of a nucleotide into a DNA strand

The carbons of the deoxyribose sugar are numbered

#3 carbon attached to an -OH group

#5 carbon holds the phosphate molecule of that nucleotide

#3 ready to bond with another nucleotide to form a polynucleotide link (5’ to3’)

Notice complimentary strand in opposite direction (5’ to 3’)

DNA always grows 5’ to 3’ never 3’ to 5’

Antiparallel Arrangement of Double Strands

Definitions Origins of Replication – where

replication of the DNA molecule begins Bacteria – circular DNA – 1 origin of

replication (RF) Eukaryotes – multiple origins of

replication (ORFS)• ORF = Replication Fork

More Definitions DNA Polymerases – enzymes that catalyze

DNA replication Leading Strand – Synthesized

continuously towards the replication fork by the DNA polymerase in one long fashion

Lagging Strand – Synthesized by short fragments away from the replication fork by the DNA polymerase

Definitions Cont’ Ligase – combines (joins) short fragments Primer – starts replication of DNA (in this

case it’s RNA) Primase – an enzyme that joins the RNA

nucleotides to make the primer Helicase – an enzyme that untwists the

double helix at the replication fork Nuclease – a DNA cutting enzyme

DNA REPLICATION -VIDEO

16-10-DNAReplication.swf

Figure 16.13 Synthesis of leading and lagging strands during DNA replication

Figure 16.14 Priming DNA synthesis with RNA

Figure 16.15 The main proteins of DNA replication and their functions

Figure 16.16 A summary of DNA replication

Figure 16.17 Nucleotide excision repair of DNA damage

A PROBLEM!

The end of the leading strand was initiated with an RNA primer Normally removed by

other DNA polymerase Removal of gaps by

DNA Polymerase doesn’t work on lagging strand end RNA primer removed

with no replacement A GAP! SHORTER AND SHORTER

FRAGMENTS?

Prokaryotes have circular DNA – no problem at ends (there aren’t ANY! Eukaryotes – have special terminal sequences of 6

nucleotides that repeat from 100-1000 times with no genes included Telomers

Protect more internal gene materials from being eroded

Germ cells / sex cells have a special enzyme (telomerase) that actually restore shortened Telomers

Somatic cells – telomer continues to shorten and may play a role in aged cell death

Cancer cells A telomerase prevents very short lengths

Figure 16.19a Telomeres and telomerase: Telomeres of mouse chromosomes

Ribonucleic Acid (RNA) Structure of RNA

Nucleotide monomer• Phosphate• Pentose sugar = ribose (extra oxygen)• Nitrogenous base (A/G/C/U)• Single stranded• 3 types (mRNA, tRNA, rRNA)

Synthesis of RNA - transcription DNA acts as a template, but only one strand of

DNA utilized at a given time This exposed strand is controlled by specific

enzymes that pair the DNA nucleotides with free RNA nucleotides which are also present in the nucleus

These RNA nucleotides form a single stranded RNA nucleic acid

DNA = ATTGGCT RNA = UAACCGA Short segments of DNA are transcribed at a time

with start and stop messages

Figure 17.2 Overview: the roles of transcription and translation in the flow of genetic information (Layer 1)

Figure 17.6 The stages of transcription: initiation, elongation, and termination (Layer 1)

Figure 17.6 The stages of transcription: elongation

Three types of RNA mRNA : messenger RNA

Transcribed from a specific segment of DNA which represents a specific gene or genetic unit

tRNA : transfer RNA Transcribed from different segments of DNA and

their function is to find a specific amino acid in the cytoplasm and bring it to the mRNA

rRNA : ribosomal RNA Transcribed at the nucleolus - with proteins

function as the site of protein synthesis

Three types of RNA

Protein Synthesis = Translation Ribosomes = sites of protein synthesis

30% - 40% protein 60% - 70% RNA (rRNA) Assembled in nucleus and exported via nuclear

pores Antibiotics can paralyze bacterial ribosomes, but

not eukaryotic ribosomes (not targeting them) 2 ribosomal subunits –a large and a small Small subunit has been used as a means of

classifying different bacteria and different invertebrates (16S)• Eukaryotes – 18S

There are three sites on the ribosome that are involved in protein synthesis

Ribosomes bring mRNA together with amino acid bearing tRNA’s

Three ribosomal sites P Site (peptidyl-tRNA) holds the tRNA carrying

the growing peptide chain after several amino acids have been added

A site (aminoacyl-tRNA) holds the next single amino acid to be added to the chain

E site (exit site) site where discharged tRNA minus amino acids leave ribosome

Figure 17.15 Translation – the basic concept

Preparation of Eukaryotic mRNA

RNA splicing- a cut and paste job to remove nucleotides from transcribed mRNA 8000 nucleotides transcribed but the

average gene contains 1200+ nucleotides

Long non-coding segments (introns) interspersed between coding segments (exons) expressed via amino acids

Figure 17.17 The initiation of translation

Figure 17.18 The elongation cycle of translation

Protein Synthesis (cont’)Initiation – elongation - termination

Starting at one end of the mRNA, the small ribosomal subunit associates with the mRNA and accepts the first tRNA with its activated amino acid attached = Initiation

tRNA associate with a triplet codon exposed on the mRNA – these are 3 nitrogenous bases that bond with 3 complementary bases exposed (anticodon) on the tRNA opposite the attached amino acid

Wobble Aren’t 61 tRNAs, are 54tRNAs

Figure 17.4 The dictionary of the genetic code

Figure 17.3 The triplet code

tRNA complexes with its amino acid in the cytoplasm using ATP – activated tRNA

The activated tRNA-amino acid complex moves towards the ribosomal area and finds a triplet codon exposed that is complementary to the anticodon of the tRNA

The first activated tRNA-amino acid, after its anticodon is bound to the mRNA codon, associates with the large ribosomal subunit which now joins the smaller subunit and the mRNA and the tRNA (TAKE A BREATH!)

The first tRNA and its amino acid now occupy the P site of the large ribosomal subunit

Review – at this point the 2 part ribosome is assembled, the mRNA has started to be read, and one tRNA plus amino acid is occupying the P site

That means the adjacent A site is free to accept a second activated tRNA and its amino acid, but only if the anticodon of this tRNA matches the next three base pairs exposed (codon)

Protein Synthesis (continued)

At this point, there are 2 tRNA-amino acid complexes adjacent to each other – Elongation involved one amino acid being added in a three step process: Codon recognition – the mRNA codon in the A

site matches with the anticodon of the tRNA –amino acid complex

Peptide bond formation between the new amino acid in the A site and the amino acid (later peptide) in the P site

Translocation

Translocation – the ribosome moves the tRNA into the A site, and its attached peptide to the P site, as the previous tRNA from the P site moves to the E (Exit) site and leaves the ribosome

Review: once this process is under way, an activated tRNA with its amino acid finds an exposed codon in the A site, attaches via H-bonds, then forms a peptide bond with the polypeptide associated with the tRNA sitting in the adjacent P site. For a moment, the longer polypeptide chain is only attached to the tRNA in the A site. Now the entire ribosome shifts so that the………

Yet More Protein Synthesis The empty tRNA from the P site

moves in to the E site and leaves the ribosome

As the tRNA with the polypeptide chain moves from the A site to the now empty P site ….exposing a new codon. GUESS WHAT HAPPENS NEXT?!

A question? Every time a new codon is exposed in the

A site, a specific tRNA-AA complex moves into the site. What originally determined this mRNA Codon?

The Answer! The original DNA that was transcribed This elongation of 1 AA takes about 0.1 s Termination – the above continues

(dozens to hundreds or more AA added) until the STOP CODON is reached (codon at the end of the mRNA)

This codon does not have a matching tRNA anticodon so the tRNA-AA attaches in the A site and the tRNA moves to the E site and releases the polypeptide chain

FINALLY - SUMMARY The take home message:

At the ribosome, the genetic language of DNA is translated into a different language – Via RNA – into the functioning language of PROTEINS!!!!

Figure 17.17 The initiation of translation

Figure 17.18 The elongation cycle of translation

Figure 17.19 The termination of translation

Figure 17.20 Polyribosomes

Table 17.1 Types of RNA in a Eukaryotic Cell

Figure 17.23 The molecular basis of sickle-cell disease: a point mutation

Figure 17.24 Categories and consequences of point mutations: Base-pair insertion or deletion

Figure 17.24 Categories and consequences of point mutations: Base-pair substitution

Figure 17.25 A summary of transcription and translation in a eukaryotic cell

Figure 18.19 Regulation of a metabolic pathway

Control of Protein SynthesisRegulation of Gene Expression

Every cell has the same numbers and types of chromosomes

Development and normal gene function requires precise gene expression in an on and off manner

Operon – cluster of gene segments on DNA and its controlling segments Repressible Inducible

Regions of the Operon (DNA)Regions of the Operon (DNA)

Promoter region : promotes transcription by binding with RNA polymerase

Operator region : binds a regulatory protein or chemical Overlaps with the RNA polymerase

binding site Structural genes : code for a particular

peptide or several peptides Start or stop codes

Figure 18.20a The trp operon: REPRESSIBLE

Figure 18.21a The lac operon: INDUCIBLE

Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells

5 DNA RNA Protein Synthesis

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