Lec12+notes

Lec12+notes - Restriction-modification systems How do...

Info iconThis preview shows pages 1–6. Sign up to view the full content.

View Full Document Right Arrow Icon
Lecture 12: Restriction, Modification, Transcription 1 Restriction-modification systems How do proteins recognize DNA? General binding activity vs sequence specific binding Where is the sequence information? 4 bases, 3 faces Transcription: chemistry, RNA polymerases Sigma factor, rho factor, rho-independent termination
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Lecture 12: Restriction, Modification, Transcription 2 Restriction and Modification • What is restriction? • Old observation: lambda phage grown on E coli B could not form plaques on E. coli K very well. For example a dilution able to make 10 6 plaques on strain B could make 10 plaques on strain K. If one of these 10 plaques were grown up and virus stocks prepared, it was now observed that the phage could form 10 6 plaques on K, but only 10 on B. One could go back and forth in this way. • Stocks were said to be restricted for growth on one strain, but modified for growth on the strain they were originally grown in. • Restriction/modification phenomena were investigated in molecular terms. • It was discovered that restriction involves cleaving DNA with a certain sequence by restriction endonucleases . • Modification involves methylating that sequence by restriction methylases , so that it cannot be cleaved by the endonuclease.
Background image of page 2
Lecture 12: Restriction, Modification, Transcription 3 Restriction endonucleases . • Enzymes often recognize palindromes of specific sequence and cut at unique sites in or near the recognition sequence. • Palindrome can be read same forward and back: Examples: Sex at noon taxes Able was I ere I saw Elba • Different enzymes recognize different sequences. Examples: GAATTC= EcoRI GATATC= EcoRV AAGCTT= Hind III • exquisite ability to recognize and cut DNA with high specificity for their recognition sequence.
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Lecture 12: Restriction, Modification, Transcription 4 • When a restriction endonuclease is expressed, a corresponding restriction methylase is also expressed that protects the host DNA from being cut. • Methylases also have high sequence specificity. • Carry out base specific methylation reactions using S adenosylmethionine as a methyl group donor. • Methylation blocks cleavage by the cognate restiction endonuclease. • Cleavage is blocked because the methyl group prevents binding of the endonuclease Restriction methylases
Background image of page 4
Modification, Transcription 5 How are specific DNA sequences recognized? General considerations in the study of nucleic acid binding proteins. • Equilibrium between binding and dissociation reactions determines occupancy of a binding site. • DNA and RNA binding proteins have a general affinity for nucleic acids and a higher affinity for the specific sequence or structure they recognize. • There are now a large number of
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 6
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 01/17/2011 for the course BIOC 100A taught by Professor Harrynoller during the Fall '10 term at UCSC.

Page1 / 32

Lec12+notes - Restriction-modification systems How do...

This preview shows document pages 1 - 6. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online