Chapter 12 and 13: Transcription and Translation

Lecture 12 October 28, 2003What’s due?

CH6 and CH10 problem set (if you haven’t all ready turned it in)

CH 11 problem set

Review: Molecular Basis of Genetics, so far…Structure

•DNA as the genetic material

*Griffith – “transforming principle”

*Avery, MacLeod and McCarty - DNA was the “transforming principle”*Hershey and Chase - DNA was the genetic material

*Composed of nucleotides –

deoxyribose phosphate group nitrogenous base*Strands are antiparallel and complementary

A – T

C - G

•Structural analysis of DNA

Review: Molecular Basis of Genetics, so far…Replication

•Mode of DNA Replication

•Semiconservative - each DNA molecule consists of one parental and one newly synthesized strand

*Meselson and Stahl – “heavy” and “light” nitrogen isotopes

•Origin of replication

•Bi-directional

•Roles of each polymerase (prokaryotes):

DNA polymerase I - primer removal, gap-filling synthesis DNA polymerase II - DNA repair DNA polymerase III - main replication enzyme

•At least six DNA polymerases in eukaryotes

Review: A Coherent Model of DNA Replication

•Helicases unwind helix (DnaA, B and C) •SSBPs prevent closure •DNA gyrase reduces tension •Association of core polymerase with template •Primase synthesizes short RNA primer •DNA synthesis (DNA pol III) •Primer removal and replacement with DNA (DNA pol I) •Ligase closes up the gaps b/w Okazaki fragments

Gene – unit of inheritance which occupies a specific chromosomal location

Gene Expression: Transcription and TranslationGene expression – mechanism by which hereditary factors are coded for and expressed (“to cause a gene to manifest its effects in the phenotype” or “the detectable effect of a gene”)

KSM: A DNA sequence that produces a functional RNA molecule

TEXT: A DNA sequence coding for a single polypeptide

Also...

Type of RNA Encodes Copies/genome

mRNA Functional protein Single or few

tRNA Molecule needed for translation

Few

rRNA Component of ribosomes Many*Non (protein) coding RNA’s

Gene Expression

Protein coding gene - A DNA sequence coding for a single polypeptide

Gene expression – mechanism by which hereditary factors are coded for and expressed

*Transcription – transfer of genetic information from DNA via synthesis of RNA

*Translation– the formation of a protein, directed by an mRNA in association with a ribosome

Genes control inherited variation via:

DNA, RNA and protein

Phenotype

Gene: A Molecular Description

5’ 3’5’3’

RNA Transcript

+1 start site terminus

Coding Region

Coding region – contains nucleotide sequence that encodes a specific protein product (this region will be translated)

Non-coding regions – contains nucleotide sequence that will get transcribed BUT not translated

In eukaryotes: introns and exons

*Un-translated regions (UTR’s)

5’ UTR 3’ UTR

Regulatory regions – sequence involved in the control of expression of a given gene, usually involves interaction with another molecule

Promoter regions – sequence involved in the control of expression of a given gene, site where RNA polymerase binds

Promoter

Only one of the two strands encodes the mRNA for a given gene

Template strand – coding strand – sense strand = template for transcription

Non-template strand – nonsense strand = RNA transcript is exactly the same as the non-sense strand

Gene: A Molecular Description

A A A G T C C G G T A C G

T T T C A G G C C A T G C

5’ 3’

3’ 5’

Given that RNA polymerase synthesizes RNA in a 5’ to 3’ direction, which strand is the template strand?

Coding strand

U U U C A G G C C A U G C3’ 5’

*Transcript will always “look” like the non-sense strand

TranscriptionTranscription – the process by which RNA molecules are synthesized on a DNA template

*RNA polymerase – enzyme that copies template strand to build an RNA molecule

reminder: RNA contains ribose, phosphate group and A, C, G and U (not T)

-synthesis in 5’ to 3’ direction –nucleotides added to 3’-OH –growing strand has base complementarity to template strand –unlike DNA pol, no primer required

*RNA polymerase (from E. coli )

2 ’

Sigma factor

Sigma factor – helps drive the polymerase to the promotor

Core

Core – responsible for elongation

Holoenzyme

Holoenzyme responsible for initiation = binding of the polymerase to the promotor

TranscriptionFactor – something that cycles on and off core complexesMultiple types of sigma factors in bacterial cells - regulation

P3

P2

P1

1

2

Promotors - sequence involved in the control of expression of a given gene, site where RNA polymerase binds

Serve three different functions:

1. ON/OFF switch 2. “Speed” switch 3. Alignment

5’ 3’5’3’ Coding

Region

RNA Transcript+1 start site

TTGACA

-35 region

TATAAT

-10, TATA box, Pribnow box~17 base spacer

Transcription in Eukaryotes*RNA polymerases:

RNA polymerase I – rRNA (18S, 28S) RNA polymerase II – mRNA RNA polymerase III – small RNA’s ( tRNA, 5S

rRNA, snRNA’s)Eukaryotic promotors:

Goldberg-Hogness box, TATA box, -25 (all)

Hogness box

Hogness box

Hogness box

Hogness box

CAAT box, -80 (many)

CAAT box

CAAT box

CAAT box

Enhancers

Transcription in Eukaryotes“Generalized Transcription Factors”–group of proteins that bind the -25 regionTranscription Factor for RNA polymerase II – TFIIA, TFIIB,

etc.*TFII’s – not enough! Need factors that bind -80 and enhancers

1. ON/OFF switch = -25 region 2. “Speed” switch = enhancers

Elongation – very similar in prokaryotes and eukaryotes

Termination -Transcription stops - Polymerase and RNA are released from DNA - DNA rehybdridizes

RNA processing in EukaryotesImmature RNA – mature RNA

*Addition of a cap at 5’ end- guanyltransferase – makes mRNA more stable, required for translation

*Addition of a poly A tail – poly A polymerase – mRNA stability, translation*Introns spliced out by spliceosome machinery