Review: Proteins and their function in the early stages of replication 1 = initiator proteins 2 = single strand binding proteins 3 = helicase 4 = 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 leading strand, 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 reading activity This is a 3’  5’ exonuclease activity DNA Polymerase activity

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

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 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 Uracil Guanine Cytosine Cytosine Guanine Thymine 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 enzyme RNA polymerase

Single RNA polymerase (Prokaryotes) Core enzyme Holoenzyme 2 ,1  and 1  ’ subunits2 , 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 transcription Recognized by RNA polymerase - 10 bases from initiation of transcription Unwinding 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

hill.com/sites/ /student_view0/chapter12/animation_ quiz_1.html Animation on Transcription