1 2 3 4 review: proteins and their function in the early stages of replication 1 = initiator...

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1 = initiator proteins2 = single strand binding proteins3 = helicase4 = topoisomerase (gyrase)

Replication

• Two DNA polymerase enzymes are necessary for replication in E. coli– DNA polymerase I – DNA polymerase III

- Along each template DNA strand, leading and lagging strands can be observed.

- The names were suggested based on synthesis at any given region.

- At any particular point in the DNA strand, if there is a leadingstrand, the complementary strand will have lagging strand.

Replication

• Two DNA polymerase enzymes are necessary for replication in E. coli– DNA polymerase I – DNA polymerase III

• Both have polymerase and exonuclease activities (functions)

• First let us take a look at the polymerase activity aspect of DNA polymerases and then discuss exonuclease activities

Replication• DNA Polymerase III

– Synthesize new DNA in the 5’ 3’ direction• Synthesizes long sequences of new DNA• Is highly processive; synthesizes DNA for a long period of time

without releasing the template• For example, synthesizes leading strand

• DNA Polymerase I– Synthesize new DNA in the 5’ 3’ direction

• Only synthesizes short sequences of new DNA• But before it could do this, it needs to remove RNA primers• This is achieved by its 5’ 3’ exonuclease activity

5’ 3’ exonuclease activity of DNA polymerase I

Replication

• The phosphodiester backbone of adjacent DNA fragments must be joined after DNA synthesis by DNA polymerases I and III

• This is done by the enzyme DNA ligase

Both DNA polymerases have proof readingactivity

This is a 3’ 5’ exonuclease activity

DNA Polymeraseactivity

Replication

• DNA Polymerase I– Synthesize new DNA in the 5’ 3’ direction

• Only synthesizes short sequences of new DNA

– 3’ 5’ exonuclease activity (proofreading)– 5’ 3’ exonuclease activity (remove primers)

• DNA Polymerase III– Synthesize new DNA in the 5’ 3’ direction

• Synthesizes long sequences of new DNA

– 3’ 5’ exonuclease activity (proofreading)

NOTE: DNA polymerase III does not have the 5’ 3’ exonuclease activity

This week we will complete…

Chapter 13 (transcription)Pages 348 – 361

Chapter 15 (translation)Pages 409 - 421

The Central Dogma(Francis Crick, 1958)

(Transcription) (Translation)

DNA RNA Protein(Gene/Genotype) (Phenotype)

An informational process between the genetic material (genotype) and the protein (phenotype)

Properties of RNA

RNA has the sugar ribose rather than deoxyribose

Properties of RNA

Nucleotides carry the bases adenine, guanine and cytosine (like DNA)

But uracil is found in place of thymine

Structure of RNA

• Designate the Nucleotides – Purines

• Guanine = G• Adenine = A

– Pyrimidines• Uracil = U• Cytosine = C

A phosphodiester bond

A phosphodiester bond

Structure of RNA

Nucleotides join together, forming a polynucleotide chain, by phosphodiester bonds

Usually single-stranded

Can have a much greater variety of complex three dimensional shapes than double-stranded DNA

Classes of RNA for Transcription and

Translation• Informational RNA (intermediate in the process of

decoding genes into polypeptides)– Messenger RNA (mRNA)

• Functional RNAs (never translated into proteins, serve other roles)– Transfer RNAs (tRNA)

• Transport amino acids to mRNA and new protein– Ribosomal RNAs (rRNA)

• Combine with an array of proteins to form ribosomes; platform for protein synthesis

– Small nuclear RNAs (snRNA)• Take part in the splicing of primary transcripts in

eukaryotes– Small cytoplasmic RNAs (scRNA)

• Direct protein traffic in eukaryotic cells– Micro RNAs (miRNA)

• Inhibits translation and induces degradation of complementary mRNA

RNA nucleotide sequences are complementary to DNA molecules

DNA template

New RNA is synthesized 5’ to 3’ and antiparallel to the template

DNA template Complementary RNA

Adenine UracilGuanine CytosineCytosine GuanineThymine Adenine

Synthesized 5’ to 3’ and antiparallel to the template

Only one strand of the DNA acts as a template for transcription

The template strand can be different for different genes

But….

For each gene only one strand of DNA serve as a template

Transcription

Catalyzed by the enzymeRNA polymerase

Single RNA polymerase (Prokaryotes)

Core enzyme Holoenzyme2 ,1 and 1 ’ subunits 2 , 1 , 1 ’ subunits plus

σ subunit

Polymerizes RNA Finds initiation sites

Initiation: The region that signals the initiation of transcription is a promoter

- 35 bases from initiation of transcriptionRecognized by RNA polymerase

- 10 bases from initiation of transcriptionUnwinding of DNA double helix begins here

Elongation: RNA is polymerized in 5’ 3’ direction

Elongation

NTPS (ATP, GTP, CTP, UTP) are added

The energy is derived by splitting the high-energy triphosphate bond

Termination

RNA polymerase recognizes signals (sequence) for chain termination

Releases the RNA and enzyme from the template

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter12/animation_quiz_1.html

Animation on Transcription