Lecture 16

Textbook Notes

16.1 What Do Genes Do?

• null alleles/loss of function alleles — do not function at all

  • Beadle and Tatum created null mutant alleles and analyzed their effects

• one-gene, one-enzyme hypothesis — one gene codes specifically for one enzyme

• genetic screen — technique for picking out particular types of mutants out of randomly generated ones

• genes contain info for all proteins produced by an organism, not just enzyme

  • one-gene, one-polypeptide hypothesis

16.2 The Central Dogma

• messenger RNA (mRNA) — carry information out of the nucleus from DNA to the site of protein synthesis in the cytoplasm

• RNA polymerase — uses a DNA template strand to polymerize ribonucleotides into strands of RNA

  • does not require a primer

• central dogma: DNA → RNA → proteins

• transcription — using a DNA template to make an RNA molecule by RNA polymerase

• translation — using the info in the base sequence of mRNA to synthesize proteins by ribosomes

• DNA (information storage) -(transcription)→ mRNA (information carrier) -(translation)→ proteins (active cell machinery)

• many genes code for RNA molecules that are not mRNA and do no get translated into proteins

17.1 An Overview of Transcription

• coding strand — the non-template strand matches the RNA sequence that is transcribed

• initiation — the stage during which RNA polymerase and other proteins assemble at the promoter sequence and open the strands of DNA to start transcription

• holoenzyme — core enzyme and additional proteins

• promoters — regions of DNA that promote the start of transcription

  • 40-50 base pairs long, similar to TATAAT → the -10 box

• downstream — DNA that is located in the direction RNA polymerase moves during trancription

  • upstream — DNA located in the opposite direction

  • the -10 box is centered about 10 bases upstream from the transcription start site

• elongation — once RNA polymerase leaves the promoter region, it catalyzes the addition of nucleotides to the 3’ end of the growing RNA, proofreading along the way

• termination — ends transcription

  • in bacteria, transcription stops when RNA polymerase transcribes a DNA sequence called a transcription-termination signal

Transcription in Eukaryotes

• eukaryotes have 3 major polymerases — pol I, II, and III

  • polymerase II creates mRNAs

• promoters are larger and more diverse (TATA box)

• general transcription factors — recognize promoters, and then RNA polymerase follows

• poly signal — RNA downstream of the poly(A) signal is cut by an enzyme

Lecture Slides

• DNA → RNA → Protein

• why the extra step?

  • amplification: most genes are present in 1 or possibly 2 copies in a cell; very little template for making proteins

  • process of making RNA from DNA template is transcription

Transcription

• carried out by DNA-dependent, RNA-synthesizing enzyme called RNA polymerase

• occurs in 4 basic steps:

  • promoter recognition

  • initiation

  • elongation

  • termination

• E. coli RNA polymerase — copies DNA into RNA

  • consists of 6 subunits (together called holoenzyme)

    • α2ββ’ωσ (without the σ subunit is called core enzyme)

  • so the holoenzyme has all 6 subunits, σ is detachable

• doesn’t transcribe the entire genome into RNA

  • a small amount of bacterial DNA and some eukaryotic DNA is not template for proteins/RNA

  • looks for individual genes (regions of useful information)

• markers (“start signal” for beginning of genes = promoters

Promoter Sequences

• -10 and -35 sites are conserved (very similar across genes) and have consensus sequence (more likely to find that base at that region than anything else)

  • -35 consensus sequence: TTGACA

  • -10 consensus sequence: TATAAT

  • the closer a bacterial promoter is to the -10 and -35 consensus sequences, the better RNA polymerase will bind = more RNA

• RNA polymerase σ subunit scanes DNA looking for promoter sites

Steps in bacterial transcription

1. nonspecific binding of polymerase holoenzyme and migration to the promoter

2. formation of a closed-promoter complex

3. formation of an open-promoter complex (DNA opens)

4. initiation of mRNA synthesis, almost always with a purine

5. elongation of mRNA by about 8 more nucleotides

6. release of σ subunit as polymerase proceeds down the template downstream from the promoter

transcription bubble

• coding strand is similar to transcript (coding strand has deoxyribose instead of ribose, T instead of U)

• template is complementary and antiparallel to transcript

• template strand is the non-coding strand

• because the RNA is synthesized 5’ → 3’, the template strand is 3’ → 5’

• how does RNA polymerase know which strand to use as the template strand?

  • either strand can be used as the template for a given gene; which strand is chosen depends on the location and orientation of the promoter sequence

  • the promoter orients RNA polymerase, determining the direction of transcription

  • the direction of transcription dictates which strand of DNA is used as the template