Gene Expression

1. Gene Expression

2. Polypeptides and Proteins • All of the work in the cell: energy generation, synthesis of new components, response to environmental stimuli, etc., is performed by proteins. • Proteins are primarily composed of polypeptides: linear chains of amino acids. • each polypeptide is called a “subunit”. • each gene produces one type of polypeptide • some proteins are composed of a single polypeptide, while others have 2 or more (up to maybe 20 in very complex proteins) subunits • Proteins, especially enzymes, also often have co-factors bound to them. • some co-factors are just metal ions such as Zn+2 • others are more complex: most of the human “vitamins” are enzyme co-factors • in many species of bacteria, co-factors are synthesized by complex pathways. • Some proteins have sugars, lipids, or other small molecules attached to them.

3. Gene Expression • Most genes code for polypeptides • maybe 5% of genes produce special RNA molecules only • The general process of gene expression is to transcribe an RNA copy of the gene, called messenger RNA (mRNA). The mRNA is then translated into the polypeptide by the action of ribosomes. • this is sometimes referred to as the Central Dogma of Molecular Biology

4. Genes on the Chromosome • The bacterial chromosome is a very long molecule of DNA. • The genes are short regions of this molecule. • A typical bacterial genome has 2000-5000 genes. • the position of each gene on the chromosome is the same in all members of a species • but not necessarily conserved across species lines • there is some local clustering of genes in the same biochemical pathway, but in general position on the chromosome cannot be correlated with gene function. • Genes have a particular orientation on the DNA strand: they are written with the 5’ end on the left and the 3’ end on the right. • However, either strand of the DNA can encode a gene. The result of this is that genes on one strand face in one direction, and genes one the other strand face the other direction

5. Transcription • Gene expression begins when the enzyme RNA polymerase binds to the promoter region just upstream from the gene. • the promoter consists of 2 segments of important nucleotides, with spacers (whose sequence doesn’t matter) in between. • positioned -10 and -35 bp upstream from RNA start • it’s a consensus sequence: variations on a common theme. • Transcription is said to start at the 5’ end of the mRNA and end at the 3’ end. This refers to the free ends of the ribose sugar in the RNA molecule. • The RNA polymerase then moves down the DNA, using one DNA strand as a template to synthesize an RNA copy of the gene. • the raw materials are “NTPs”: nucleoside triphosphates. The energy needed to do the synthesis come from removing the 2 terminal phosphate groups, the same process as in using ATP energy. • Transcription ends at a terminator sequence. The RNA polymerase falls off the DNA, releasing the new mRNA. • In eukaryotes, the mRNA is processed by splicing out introns and protecting the ends. These events do not occur in prokaryotes.

6. Transcription Control • The key event is binding of RNA polymerase to the promoter. It is affected by several factors. • promoters vary slightly in sequence, and these variations affect the strength of binding. • RNA polymerase has a subunit called sigma. There are several different sigma factors in the cell, each of which is specific to a different class of promoter. This provides large-scale gene regulation. • genes are controlled individually by the binding of regulatory proteins, called transcription factors, to regions near the promoter. • Some transcription factors block transcription and others encourage it • Some transcription factors regulate whole groups of genes scattered throughout the chromosome: a regulon • Lac operon model (Jacob and Monod). • Gene used to convert the sugar lactose into glucose, which is used as food. • The repressor protein (transcription factor) binds to DNA (the operator) near the promoter and physically blocks transcription. • when lactose is present, the repressor binds to it, changes its conformation, and falls off the DNA. This allows RNA polymerase to bind and transcription to proceed.

7. Transcription Termination • Two basic types: rho-dependent and rho-independent. • rho is a protein • Rho-dependent termination involves rho protein binding to the RNA and moving along it until it catches up with the RNA polymerase and knocks it off the DNA • Rho-independent termination involves the newly synthesized RNA folding up into a hairpin loop, due to complementary bases. This sudden folding knocks the RNA polymerase off the DNA. • formation of stem-loops in RNA also affects transcription initiation in some genes

8. mRNA, Translation, Operons • Translation is the process of converting the information on messenger RN.A into protein. • Note that not all of the mRNA is translated: there is an untranslated region of variable length at both ends, called 5’-UTR and 3’-UTR • In bacteria, some adjacent genes with related functions are transcribed onto the same mRNA. This is called an operon. • the proteins are translated individually • this allows a fixed ratio of the proteins to be produced.

9. Genetic Code • There are only 4 bases in DNA and RNA, but there are 20 different amino acids that go into proteins. How can DNA code for the amino acid sequence of a protein? • Each amino acid is coded for by a group of 3 bases, a codon. 3 bases of DNA or RNA = 1 codon. • Since there are 4 bases and 3 positions in each codon, there are 4 x 4 x 4 = 64 possible codons. • This is far more than is necessary, so most amino acids use more than 1 codon. • 3 of the 64 codons are used as STOP signals; they are found at the end of every gene and mark the end of the protein. • In bacteria, translation usually begins at an ATG codon, but GTG and TTG are also common start codons. • and, a few others are occasionally used • In all cases, the first amino acid is N-formyl methionine, a derivative of the normal methionine used within the protein. • note that “start” codons are also used within the protein • this makes is difficult to be sure where the protein actually starts

10. Transfer RNA • Transfer RNA molecules act as adapters between the codons on messenger RNA and the amino acids. Transfer RNA is the physical manifestation of the genetic code. • Each transfer RNA molecule is twisted into a knot that has 2 ends. • At one end is the “anticodon”, 3 RNA bases that matches the 3 bases of the codon. This is the end that attaches to messenger RNA. • At the other end is an attachment site for the proper amino acid. • A special group of enzymes (aminoacyl tRNA synthetases, which are highly conserved in evolution) pairs up the proper transfer RNA molecules with their corresponding amino acids. • Transfer RNA brings the amino acids to the ribosomes, which are RNA/protein hybrids that move along the messenger RNA, translating the codons into the amino acid sequence of the polypeptide.

11. Translation • Three main players here: messenger RNA, the ribosome, and the transfer RNAs with attached amino acids. • First step: initiation. The messenger RNA binds to a ribosome, and the transfer RNA corresponding to the START codon binds to this complex. Ribosomes are composed of 2 subunits (large and small), which come together when the messenger RNA attaches during the initiation process. • there are also several protein “initiation factors” that assist in this process

12. More Translation • Step 2 is elongation: the ribosome moves down the messenger RNA, adding new amino acids to the growing polypeptide chain. • The ribosome has 2 sites for binding transfer RNA. The first RNA with its attached amino acid binds to the first site, and then the transfer RNA corresponding to the second codon bind to the second site. • The ribosome then removes the amino acid from the first transfer RNA and attaches it to the second amino acid. • At this point, the first transfer RNA is empty: no attached amino acid, and the second transfer RNA has a chain of 2 amino acids attached to it.

13. Translation, part 3 • The ribosome then slides down the messenger RNA 1 codon (3 bases). • The first transfer RNA is pushed off, and the second transfer RNA, with 2 attached amino acids, moves to the first position on the ribosome.

14. Translation, part 4 • The elongation cycle repeats as the ribosome moves down the messenger RNA, translating it one codon and one amino acid at a time. • Repeat until a STOP codon is reached.

15. Translation, end • The final step in translation is termination. When the ribosome reaches a STOP codon, there is no corresponding transfer RNA. • Instead, a small protein called a “release factor” attaches to the stop codon. • The release factor causes the whole complex to fall apart: messenger RNA, the two ribosome subunits, the new polypeptide. • The messenger RNA can be translated many times, to produce many protein copies.

16. Post-translation • The new polypeptide is now floating loose in the cytoplasm. It might also be inserted into a membrane, if the ribosome it was translated on was attached to the membrane by a special RNA/protein hybrid molecule. • Polypeptides fold spontaneously into their active configuration, and they spontaneously join with other polypeptides to form the final proteins. • Sometimes other molecules are also attached to the polypeptides: sugars, lipids, phosphates, etc. All of these have special purposes for protein function.