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

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 were 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 Crick

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

# 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

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

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